Humanized anti-CXCR5 antibodies, derivatives thereof and their use

ABSTRACT

The present invention relates to humanized antibodies that specifically bind to CXCR5 and can, for example, inhibit CXCR5 function. The invention also includes uses of the antibodies to treat or prevent CXCR5 related diseases or disorders.

This application is a division of U.S. application Ser. No. 12/675,799,filed Oct. 25, 2010, which was a national stage application under 35U.S.C. § 371 of International Application No. PCT/US2008/074381, filedAug. 27, 2008, which claims the benefit of U.S. Provisional ApplicationNo. 60/968,792, filed Aug. 29, 2007, the disclosures of each of whichare explicitly incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to anti-CXCR5 antibodies and their use inthe amelioration, treatment or prevention of diseases or disorders inmammals, including humans, resulting from improper CXCR5 activity ormetabolism, or the inappropriate or adventitious use thereof, forexample, by a pathogen. An antibody of interest may block engagement ofa ligand, such as CXCL13, with it receptor, such as, CXCR5.Prophylactic, immunotherapeutic and diagnostic compositions comprisingthe antibodies and derivatives thereof of interest and their use inmethods for preventing or treating diseases in mammals, includinghumans, caused by inappropriate metabolism and/or activity of CXCR5⁺cells, such as B lymphocytes, also are disclosed. Such diseases includeautoimmune deficiencies and diseases caused by or characterized byinflammation, such as rheumatoid arthritis (RA), where CXCR5 isup-regulated.

BACKGROUND

CXCR5, also known as Burkitt lymphoma receptor (BLR1), CD185, MDR15 andMGC117347, is a G protein-coupled receptor which is a member of the CXCchemokine receptor family. A ligand is BLC, also known as CXCL13, whichis a B cell chemoattractant.

The unprocessed CXCR5 precursor is 372 amino acids in length with amolecular weight of 42 K_(D).

CXCR5 has a role in B cell migration and localization within particularanatomic compartments. Knockout mice lack peripheral lymph nodes, havefewer Peyer's patches and have decreased B cell levels.

SUMMARY

The present invention provides novel humanized and human antibodies, andfragments and derivatives thereof, that specifically bind to CXCR5. Someof the antibodies, and CXCR5-binding fragments thereof, can be alteredto prevent intrachain disulfide bond formation resulting in a moleculethat is stable through manufacturing and use in vivo. Other antibodiesof interest can be altered to minimize binding to F_(c)R. Some CXCR5antibodies of interest compete with CXCL13 for binding to CXCR5. Otherantibodies diminish CXCR5 activity.

The invention includes the amino acid sequences of the variable heavyand light chain of the antibodies and their corresponding nucleic acidsequences.

Another embodiment of the invention includes the complementaritydetermining regions (CDR) sequences of the antibodies to obtain bindingmolecules that comprise one or more CDR regions, or CDR-derived regions,that retain CXCR5-binding capacity of the parent molecule from which theCDR was(were) obtained.

An antibody of interest can be one that prevents CXCL13, or otherligand, binding to CXCR5⁺ cells, such as B cells.

Another embodiment of the present invention includes the cell lines andvectors harboring the antibody sequences of the present invention.

Another embodiment of the present invention is the use of the antibodiesfor the preparation of a medicament or composition for the treatment ofdiseases and disorders associated with CXCR5 function and metabolism.

Another embodiment of the present invention is the use of theseantibodies in the treatment of disorders associated with atypical orabnormal CXCR5 biology and function.

Additional features and advantages are described herein, and will beapparent from, the following Detailed Description.

DETAILED DESCRIPTION

This invention is not limited to the particular methodology, protocols,cell lines, vectors, or reagents described herein because they may varywithout departing from the spirit and scope of the invention. Further,the terminology used herein is for the purpose of exemplifyingparticular embodiments only and is not intended to limit the scope ofthe present invention. Unless defined otherwise, all technical andscientific terms and any acronyms used herein have the same meanings ascommonly understood by one of ordinary skill in the art in the field ofthe invention. Any method and material similar or equivalent to thosedescribed herein can be used in the practice of the present inventionand only exemplary methods, devices, and materials are described herein.

All patents and publications mentioned herein are incorporated herein inentirety by reference for the purpose of describing and disclosing theproteins, enzymes, vectors, host cells and methodologies reportedtherein that might be used with and in the present invention. However,nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

Prior to teaching the making and using of the CXCR5-related methods andproducts of interest, the following non-limiting definitions of someterms and phrases are provided to guide the artisan.

“CXCR5” relates to the naturally occurring, known molecule found onlymphocytes, particularly B cells, and particularly naïve B cells; tosuch a molecule isolated from such cells; to such a moleculemanufactured recombinantly using known materials and means, and using anucleic acid encoding a CXCR5; as well as to portions of CXCR5, such asthe extracellular (EC) domain, which retains the characteristics andproperties relevant to the practice of the instant invention, such asCXCL13 binding. A soluble CXCR5 molecule can consist essentially of theEC domain of CXCR5, which includes, generally, about the first sixtyamino acids of the molecule, that is, the amino terminal portion ofCXCR5.

CXCR5 is a non-promiscuous receptor. CXCL13 is a ligand of CXCR5 and isexpressed constitutively on stromal cells, such as follicular dendriticcells, and in lymphoid tissues. CXCL13 specifically attracts B cells anda small subset of T cells called B helper follicular T cells, TFH. Thatmay not be unexpected given the many interactions between T cell and Bcell populations in the immune system. Moreover, activated T cellinduces or upregulate CXCR5 expression. Infiltration of lymphocytes intotertiary, ectopic germinal centers (GCs) has been found to correlatewell with increased disease severity and tolerance breakdown in certaindisorders which preset with such atypical lymph node-like structures.Using in vivo murine models, such as CXCR5−/− and CXCL13−/− mice, theabsence of either the receptor or the ligand results in an altered GCfine architecture due to altered T and B cell localization, and possiblyinteraction. These mice are also protected against developing severecollagen-induced arthritis (CIA). As CXCR5 is selectively expressed onmature B cells, which are linked to the pathogenesis of RA, blockingthis receptor will modulate the arthritogenic response in affectedindividuals. Rheumatoid arthritis treatment with biologics (i.e.,anti-TNFα and anti-CD20 antibodies, Rituximab) has shown to beclinically effective; in particular, patients on B cell-directed therapyhave shown long-lasting improvements in clinical signs and symptoms.Selective targeting of CXCR5, which is only expressed on mature B cellsand B helper T cells, will not affect B cell development orimmunocompromise the patient. Unlike Rituximab, an instant antibody is aneutralizing antibody that does not mediate cell cytotoxicity.

A “CXCR5 disease” is a malady, disorder, disease, condition, abnormalityand so on, which is characterized by or caused by overexpression orincreased levels of CXCL13 or other CXCR5 ligand, increased levels of Bcells, increased levels of B cell activity, increased levels of CXCR5 orimproper metabolism and activity of CXCR5.

By “B cell activity” is meant higher than normal B cell levels, whichcan be local, or evidence of a biological manifestation or function of aB cell, such as antibody expression, Bruton's tyrosine kinase presenceor activity, expression or presence of CD 19, expression or presence ofB cell activating factor and so on.

The phrase “substantially identical” with respect to an antibody chainpolypeptide sequence may be construed as an antibody chain exhibiting atleast 70%, 80%, 90%, 95% or more sequence identity to the referencepolypeptide sequence. The term with respect to a nucleic acid sequencemay be construed as a sequence of nucleotides exhibiting at least about85%, 90%, 95%, 97% or more sequence identity to the reference nucleicacid sequence.

The terms, “identity” or “homology” may mean the percentage ofnucleotide bases or amino acid residues in the candidate sequence thatare identical with the residue of a corresponding sequence to which itis compared, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent identity for the entiresequence, and not considering any conservative substitutions as part ofthe sequence identity. Neither N-terminal or C-terminal extensions norinsertions shall be construed as reducing identity or homology. Methodsand computer programs for the alignment are available and well known inthe art. Sequence identity may be measured using sequence analysissoftware.

The phrases and terms “functional fragment, variant, derivative oranalog” and the like, as well as forms thereof, of an antibody orantigen is a compound or molecule having qualitative biological activityin common with a full-length antibody or antigen of interest. Forexample, a functional fragment or analog of an anti-CXCR5 antibody isone which can bind to a CXCR5 molecule or one which can prevent orsubstantially reduce the ability of a ligand, such as CXCL13, or anagonistic or antagonistic antibody, to bind to CXCR5. An example is anscF_(V) molecule. As to CXCR5, a variant or derivative thereof is amolecule that is not identical to a naturally occurring CXCR5 and yetcan be used for a purpose of the instant invention, such as, while notidentical to the wild type CXCR5 nevertheless can be used as immunogento raise antibodies that selectively bind to wild type CXCR5.

“Substitutional” variants are those that have at least one amino acidresidue in a native sequence removed and replaced with a different aminoacid inserted in its place at the same position. The substitutions maybe single, where only one amino acid in the molecule is substituted, ormay be multiple, where two or more amino acids are substituted in thesame molecule. The plural substitutions may be at consecutive sites.Also, one amino acid can be replaced with plural residues, in which casesuch a variant comprises both a substitution and an insertion.“Insertional” variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative sequence. Immediately adjacent to an amino acid means connectedto either the α-carboxyl or α-amino functional group of the amino acid.“Deletional” variants are those with one or more amino acids in thenative amino acid sequence removed. Ordinarily, deletional variants willhave one or two amino acids deleted in a particular region of themolecule.

The term “antibody” is used in the broadest sense, and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), antibody fragments or synthetic polypeptidescarrying one or more CDR or CDR-derived sequences so long as thepolypeptides exhibit the desired biological activity. Antibodies (Abs)and immunoglobulins (Igs) are glycoproteins having the same structuralcharacteristics. Generally, antibodies are considered Igs with a definedor recognized specificity. Thus, while antibodies exhibit bindingspecificity to a specific target, immunoglobulins include bothantibodies and other antibody-like molecules which lack targetspecificity. The antibodies of the invention can be of any class (e.g.,IgG, IgE, IgM, IgD, IgA and so on), or subclass (e.g., IgG₁, IgG₂,IgG_(2a), IgG₃, IgG₄, IgA₁, IgA₂ and so on) (“type” and “class”, and“subtype” and “subclass”, are used interchangeably herein). Native orwildtype, that is, obtained from a non-artificially manipulated memberof a population, antibodies and immunoglobulins are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachheavy chain has at one end a variable domain (V_(H)) followed by anumber of constant domains. Each light chain has a variable domain atone end (V_(L)) and a constant domain at the other end. By“non-artificially manipulated” is meant not treated to contain orexpress a foreign antigen binding molecule. Wildtype can refer to themost prevalent allele or species found in a population or to theantibody obtained from a non-manipulated animal, as compared to anallele or polymorphism, or a variant or derivative obtained by a form ofmanipulation, such as mutagenesis, use of recombinant methods and so onto change an amino acid of the antigen-binding molecule.

As used herein, “anti-CXCR5 antibody” means an antibody or polypeptidederived therefrom (a derivative) which binds specifically to human CXCR5as defined herein, including, but not limited to, molecules whichinhibit or substantially reduce the binding of CXCR5 to its ligands orinhibit CXCR5 activity.

The term “variable” in the context of a variable domain of antibodies,refers to certain portions of the pertinent molecule which differextensively in sequence between and among antibodies and are used in thespecific recognition and binding of a particular antibody for itsparticular target. However, the variability is not evenly distributedthrough the variable domains of antibodies. The variability isconcentrated in three segments called complementarity determiningregions (CDRs; i.e., CDR1, CDR2, and CDR3) also known as hypervariableregions, both in the light chain and the heavy chain variable domains.The more highly conserved portions of variable domains are called theframework (FR) regions or sequences. The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting aβ-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the β-sheet structure.The CDRs in each chain are held together often in proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the target (epitope or determinant) binding site ofantibodies (see Kabat et al. Sequences of Proteins of ImmunologicalInterest, National Institute of Health, Bethesda, Md. (1987)). As usedherein, numbering of immunoglobulin amino acid residues is doneaccording to the immunoglobulin amino acid residue numbering system ofKabat et al., unless otherwise indicated. One CDR can carry the abilityto bind specifically to the cognate epitope.

The term “antibody fragment” refers to a portion of an intact or afull-length chain or an antibody, generally the target binding orvariable region. Examples of antibody fragments include, but are notlimited to, F_(ab), F_(ab′, F) _((ab′)2) and F_(v) fragments. A“functional fragment” or “analog of an anti-CXCR5 antibody” is one whichcan prevent or substantially reduce the ability of the receptor to bindto a ligand or to initiate signaling. As used herein, functionalfragment generally is synonymous with, “antibody fragment” and withrespect to antibodies, can refer to fragments, such as F_(v), F_(ab),F_((ab′)2) and so on which can prevent or substantially reduce theability of the receptor to bind to a ligand or to initiate signaling. An“F_(v)” fragment consists of a dimer of one heavy and one light chainvariable domain in a non-covalent association (V_(H)-V_(L) dimer). Inthat configuration, the three CDRs of each variable domain interact todefine a target binding site on the surface of the V_(H)-V_(L) dimer, asin an intact antibody. Collectively, the six CDRs confer target bindingspecificity on the intact antibody. However, even a single variabledomain (or half of an F_(v) comprising only three CDRs specific for atarget) can have the ability to recognize and to bind target.

“Single-chain F_(v),” “sF_(v)” or “scAb” antibody fragments comprise theV_(H) and V_(L) domains of an antibody, wherein these domains arepresent in a single polypeptide chain. Generally, the F_(v) polypeptidefurther comprises a polypeptide linker, often a flexible molecule,between the V_(H) and V_(L) domains, which enables the sFv to form thedesired structure for target binding.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments can comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain. By using a linker that is tooshort to allow pairing between the two variable domains on the samechain, the diabody domains are forced to pair with the binding domainsof another chain to create two antigen-binding sites.

The F_(ab) fragment contains the variable and constant domains of thelight chain and the variable and first constant domain (C_(H1)) of theheavy chain. F_(ab′) fragments differ from F_(ab) fragments by theaddition of a few residues at the carboxyl terminus of the C_(H1) domainto include one or more cysteines from the antibody hinge region. F_(ab′)fragments can be produced by cleavage of the disulfide bond at the hingecysteines of the F_((ab′)2) pepsin digestion product. Additionalenzymatic and chemical treatments of antibodies can yield otherfunctional fragments of interest.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts.

Monoclonal antibodies herein specifically include “chimeric” antibodiesin which a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass (type or subtype), with the remainder of the chain(s) identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity of binding to CXCR5 or impacting CXCR5activity or metabolism (U.S. Pat. No. 4,816,567; and Morrison et al.,Proc Natl Acad Sci USA 81:6851 (1984)). Thus, CDRs from one class ofantibody can be grafted into the FR of an antibody of different class orsubclass.

Monoclonal antibodies are highly specific, being directed against asingle target site, epitope or determinant. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes)of an antigen, each monoclonal antibody is directed against a singledeterminant on the target. In addition to their specificity, monoclonalantibodies are advantageous being synthesized by a host cell,uncontaminated by other immunoglobulins, and provides for cloning therelevant gene and mRNA encoding the antibody of chains thereof. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies for use withthe present invention may be isolated from phage antibody librariesusing well known techniques or can be purified from a polyclonal prep.The parent monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method described byKohler et al., Nature 256:495 (1975), or may be made by recombinantmethods well known in the art.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such asF_(v), F_(ab), F_(ab′), F_((ab′)2) or other target-binding subsequencesof antibodies) which contain sequences derived from non-humanimmunoglobulin, as compared to a human antibody. In general, thehumanized antibody will comprise substantially all of one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulintemplate sequence. The humanized antibody may also comprise at least aportion of an immunoglobulin constant region (F_(c)), typically that ofthe human immunoglobulin template chosen. In general, the goal is tohave an antibody molecule that is minimally immunogenic in a human.Thus, it is possible that one or more amino acids in one or more CDRsalso can be changed to one that is less immunogenic to a human host,without substantially minimizing the specific binding function of theone or more CDRs to CXCR5 or to CXCL13. Alternatively, the FR can benon-human but those amino acids most immunogenic are replaced with onesless immunogenic. Nevertheless, CDR grafting, as discussed above, is notthe only way to obtain a humanized antibody. For example, modifying justthe CDR regions may be insufficient as it is not uncommon for frameworkresidues to have a role in determining the three-dimensional structureof the CDR loops and the overall affinity of the antibody for itsligand. Hence, any means can be practiced so that the non-human parentantibody molecule is modified to be one that is less immunogenic to ahuman, and global sequence identity with a human antibody is not alwaysa necessity. So, humanization also can be achieved, for example, by themere substitution of just a few residues, particularly those which areexposed on the antibody molecule and not buried within the molecule, andhence, not readily accessible to the host immune system. Such a methodis taught herein with respect to substituting “mobile” or “flexible”residues on the antibody molecule, the goal being to reduce or dampenthe immunogenicity of the resultant molecule without comprising thespecificity of the antibody for its epitope or determinant. See, forexample, Studnicka et al., Prot Eng 7(6)805-814, 1994; Mol Imm44:1986-1988, 2007; Sims et al., J Immunol 151:2296 (1993); Chothia etal., J Mol Biol 196:901 (1987); Carter et al., Proc Natl Acad Sci USA89:4285 (1992); Presta et al., J Immunol 151:2623 (1993), WO 2006/042333and U.S. Pat. No. 5,869,619.

The adaptive immune response has two major arms: the cellular immuneresponse of T lymphocytes and the humoral immune response of antibodysecreting B lymphocytes. B cell epitopes can be linear, contiguous aminoacids, or can be conformational (Protein Science (2005) 14, 246). Incontrast, T-cell epitopes are short linear peptides that are cleavedfrom antigenic proteins that are presented in the context of majorhistocompatibility complex (MHC) proteins, or, in case of humans, humanleukocyte antigen (HLA) class I or class II molecules. Epitopepresentation depends on both MHC-peptide binding and T cell receptor(TCR) interactions. MHC proteins are highly polymorphic, and each bindsto a limited set of peptides. Thus, the particular combination of MHCalleles present in a host limits the range of potential epitopesrecognized during an infection.

Two fundamental types of T cells are distinguished by expression of CD8and CD4 proteins, which dictate whether a T cell will recognize epitopespresented by class I or class II molecules, respectively. CD4⁺ Tepitopes are processed after encapsulation by antigen presenting cellsin membrane bound vesicles, where the antigen is degraded by proteasesinto peptide fragments that bind to MHC class II proteins. In contrast,CD8⁺ T cells generally recognize viral or self-antigens expressed fromwithin a cell, proteins that are cleaved into short peptides in thecytosol by the immunoproteasome. After cleavage, peptides aretranslocated by the transporter associated with antigen processing (TAP)into the endoplasmic reticulum for loading onto HLA I antigens. CD4⁺ T(helper) cell epitopes are critical in driving T cell-dependent immuneresponses to protein antigens.

A humanization method of interest is based on the impact of themolecular flexibility of the antibody during and at immune recognition.Protein flexibility is related to the molecular motion of the proteinmolecule. Protein flexibility is the ability of a whole protein, a partof a protein or a single amino acid residue to adopt an ensemble ofconformations which differ significantly from each other. Informationabout protein flexibility can be obtained by performing protein X-raycrystallography experiments (see, for example, Kundu et al. 2002,Biophys J 83:723-732.), nuclear magnetic resonance experiments (see, forexample, Freedberg et al., J Am Chem Soc 1998, 120(31):7916-7923) or byrunning molecular dynamics (MD) simulations. An MD simulation of aprotein is done on a computer and allows one to determine the motion ofall protein atoms over a period of time by calculating the physicalinteractions of the atoms with each other. The output of a MD simulationis the trajectory of the studied protein over the period of time of thesimulation. The trajectory is an ensemble of protein conformations, alsocalled snapshots, which are periodically sampled over the period of thesimulation, e.g. every 1 picosecond (ps). It is by analyzing theensemble of snapshots that one can quantify the flexibility of theprotein amino acid residues. Thus, a flexible residue is one whichadopts an ensemble of different conformations in the context of thepolypeptide within which that residue resides. MD methods are known inthe art, see, e.g., Brooks et al. “Proteins: A Theoretical Perspectiveof Dynamics, Structure and Thermodynamics” (Wiley, New York, 1988).Several software enable MD simulations, such as Amber (see Case et al.(2005) J Comp Chem 26:1668-1688), Charmm (sec Brooks et al. (1983) JComp Chem 4:187-217; and MacKerell et al. (1998) in “The Encyclopedia ofComputational Chemistry” vol. 1:271-177, Schleyer et al., eds.Chichester: John Wiley & Sons) or Impact (see Rizzo et al. J Am ChemSoc; 2000; 122(51):12898-12900.)

Most protein complexes share a relatively large and planar buriedsurface and it has been shown that flexibility of binding partnersprovides the origin for their plasticity, enabling them toconformationally adapt to each other (Structure (2000) 8, R137-R142). Assuch, examples of “induced fit” have been shown to play a dominant rolein protein-protein interfaces. In addition, there is a steadilyincreasing body of data showing that proteins actually bind ligands ofdiverse shapes sizes and composition (Protein Science (2002) 11:184-187)and that the conformational diversity appears to be an essentialcomponent of the ability to recognize different partners (Science (2003)299, 1362-1367). Flexible residues are involved in the binding ofprotein-protein partners (Structure (2006) 14, 683-693).

The flexible residues can adopt a variety of conformations that providean ensemble of interaction areas that are likely to be recognized bymemory B cells and to trigger an immunogenic response. Thus, antibodycan be humanized by modifying a number of residues from the framework sothat the ensemble of conformations and of recognition areas displayed bythe modified antibody resemble as much as possible those adopted by ahuman antibody.

That can be achieved by modifying a limited number of residues by: (1)building a homology model of the parent mAb and running an MDsimulation; (2) analyzing the flexible residues and identification ofthe most flexible residues of a non-human antibody molecule, as well asidentifying residues or motifs likely to be a source of heterogeneity orof degradation reaction; (3) identifying a human antibody which displaysthe most similar ensemble of recognition areas as the parent antibody;(4) determining the flexible residues to be mutated, residues or motifslikely to be a source of heterogeneity and degradation are also mutated;and (5) checking for the presence of known T cell or B cell epitopes.The flexible residues can be found using an MD calculation as taughtherein using an implicit solvent model, which accounts for theinteraction of the water solvent with the protein atoms over the periodof time of the simulation.

Once the set of flexible residues has been identified within thevariable light and heavy chains, a set of human heavy and light chainvariable region frameworks that closely resemble that of the antibody ofinterest are identified. That can be done, for example, using a blastsearch on the set of flexible residues against a database of antibodyhuman germ line sequence. It can also be done by comparing the dynamicsof the parent mAb with the dynamics of a library of germ line canonicalstructures. The CDR residues and neighboring residues are excluded fromthe search to ensure high affinity for the antigen is preserved.

Thus, a comparison the molecular dynamic trajectory of the antibody ofinterest with the trajectories of a library of germ line antibodystructures was conducted. 16D7 was compared to a library of 49 germ linestructures. The molecular dynamic trajectory retained of each antibodyis an ensemble of molecular dynamic calculations during the moleculardynamics computer simulation where, for example, about 10 diverseconformations are used as diverse starting points, and for each startingpoint, about 10 molecular dynamic simulations are run. The 49 3Dhomology models of the human antibody germ lines were built bysystematically combining the 7 most frequent human light chain (vκ1,vκ2, vκ3, vκ4, vλ1, v1λ2 and vλ3) and the 7 most frequent heavy chains(vh1a, vh1b, vh2, vh3, vh4, vh5 and vh6) (Nucleic Acids Research, 2005,Vol. 33, Database issue D593-D597). The flexible residues of 16D7 arethen changed to the corresponding residues of the germ line structurewith a trajectory closest to that of the antibody of interest.

Flexible residues then are replaced. When several human residues showsimilar homologies, the selection is driven also by the nature of theresidues that are likely to affect the solution behavior of thehumanized antibody. For instance, polar residues will be preferred inexposed flexible loops over hydrophobic residues. Residues which are apotential source of instability and heterogeneity are also mutated evenif there are found in the CDRs. That will include exposed methionines assulfoxide formation can result from oxygen radicals, proteolyticcleavage of acid labile bonds such as those of the Asp-Pro dipeptide(Drug Dev Res (2004) 61:137-154), deamidation sites found with anexposed asparagine residue followed by a small amino acid, such as Gly,Ser, Ala, His, Asn or Cys Chromatog (2006) 837:35-43) andN-glycosylation sites, such as the Asn-X-Ser/Thr site. Typically,exposed methionines will be substituted by a Leu, exposed asparagineswill be replaced by a glutamine or by an aspartate, or the subsequentresidue will be changed. For the glycosylation site (Asn-X-Ser/Thr),either the Asn or the Ser/Thr residue will be changed.

The resulting composite sequence is checked for the presence of known Bcell or linear T-cell epitopes. A search is performed, for example, withthe publicly available Immune Epitope Data Base (IEDB) (PLos Biol (2005)3(3)e91). If a known epitope is found within the composite sequence,another set of human sequences is retrieved and substituted

Unlike the resurfacing method of U.S. Pat. No. 5,639,641, bothB-cell-mediated and T-cell-mediated immunogenic responses are addressedby the method. The method also avoids the issue of loss of activity thatis sometimes observed with CDR grafting (U.S. Pat. No. 5,530,101). Inaddition, stability and solubility issues also are considered in theengineering and selection process, resulting in an antibody that isoptimized for low immunogenicity, high antigen affinity and improvedbiophysical properties.

Strategies and methods for resurfacing antibodies, and other methods forreducing immunogenicity of antibodies within a different host, aredisclosed, for example, in U.S. Pat. No. 5,639,641. Briefly, in apreferred method, (1) position alignments of a pool of antibody heavyand light chain variable regions are generated to yield heavy and lightchain variable region framework surface exposed positions, wherein thealignment positions for all variable regions are at least about 98%identical; (2) a set of heavy and light chain variable region frameworksurface exposed amino acid residues is defined for a non-human, such asa rodent antibody (or fragment thereof); (3) a set of heavy and lightchain variable region framework surface exposed amino acid residues thatis most closely identical to the set of rodent surface exposed aminoacid residues is identified; and (4) the set of heavy and light chainvariable region framework surface exposed amino acid residues defined instep (2) is substituted with the set of heavy and light chain variableregion framework surface exposed amino acid residues identified in step(3), except for those amino acid residues that are within 5 Å of anyatom of any residue of a CDR of the rodent antibody, to yield ahumanized, such as a rodent antibody retaining binding specificity.

Antibodies can be humanized by a variety of other techniques includingCDR grafting (EPO 0 239 400; WO 91/09967; and U.S. Pat. Nos. 5,530,101and 5,585,089), veneering or resurfacing (EPO 0 592 106; EPO 0 519 596;Padlan, 1991, Molec Imm 28(4/5):489-498; Studnicka et al., 1994, ProtEng 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973) and chainshuffling (U.S. Pat. No. 5,565,332). Human antibodies can be made by avariety of methods known in the art including, but not limited to, phagedisplay methods, see U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806 and5,814,318; and WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735 and WO 91/10741, using transgenic animals, such asrodents, using chimeric cells and so on.

“Antibody homolog” or “homolog” refers to any molecule whichspecifically binds CXCR5 as taught herein. Thus, an antibody homologincludes native or recombinant antibody, whether modified or not,portions of antibodies that retain the biological properties ofinterest, such as binding CXCR5, such as an F_(ab) or F_(v) molecule, asingle chain antibody, a polypeptide carrying one or more CDR regionsand so on. The amino acid sequence of the homolog need not be identicalto that of the naturally occurring antibody but can be altered ormodified to carry substitute amino acids, inserted amino acids, deletedamino acids, amino acids other than the twenty normally found inproteins and so on to obtain a polypeptide with enhanced or otherbeneficial properties.

Antibodies with homologous sequences are those antibodies with aminoacid sequences that have sequence homology with the amino acid sequenceof a CXCR5 antibody of the present invention. Preferably, homology iswith the amino acid sequence of the variable regions of an antibody ofthe present invention. “Sequence homology” as applied to an amino acidsequence herein is defined as a sequence with at least about 90%, 91%,92%, 93%, 94% or more sequence homology, and more preferably at leastabout 95%, 96%, 97%, 98% or 99% sequence homology to another amino acidsequence, as determined, for example, by the FASTA search method inaccordance with Pearson & Lipman, Proc Natl Acad Sci USA 85, 2444-2448(1988).

A chimeric antibody is one with different portions of an antibodyderived from different sources, such as different antibodies, differentclasses of antibody, different animal species, for example, an antibodyhaving a variable region derived from a murine monoclonal antibodypaired with a human immunoglobulin constant region and so on. Thus, ahumanized antibody is a species of chimeric antibody. Methods forproducing chimeric antibodies are known in the art, see, e.g., Morrison,1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies etal., 1989, J Immunol Methods 125:191-202; and U.S. Pat. Nos. 5,807,715,4,816,567, and 4,816,397.

Artificial antibodies include single chain antibodies, scFv fragments,chimeric antibodies, diabodies, triabodies, tetrabodies and mru (seereviews by Winter & Milstein, 1991, Nature 349:293-299; and Hudson,1999, Curr Opin Imm 11:548-557), each with antigen-binding orepitope-binding ability. In the single chain F_(v) fragment (scF_(v)),the V_(H) and V_(L) domains of an antibody are linked by a flexiblepeptide. Typically, the linker is a peptide of about 15 amino acids. Ifthe linker is much smaller, for example, 5 amino acids, diabodies areformed, which are bivalent scFv dimers. If the linker is reduced to lessthan three amino acid residues, trimeric and tetrameric structures areformed that are called triabodies and tetrabodies, respectively. Thesmallest binding unit of an antibody can be a single CDR, typically theCDR2 or 3 of the heavy chain which has sufficient specific recognitionand binding capacity, but can be any combination of CDR sequences as canbe determined practicing the methods taught herein. Such a fragment iscalled a molecular recognition unit or mru. Several such mrus can belinked together with short linker peptides, therefore forming anartificial binding protein with higher avidity than a single mru.

Also included within the scope of the invention are functionalequivalents of an antibody of interest. The term “functionalequivalents” includes antibodies with homologous sequences, antibodyhomologs, chimeric antibodies, artificial antibodies and modifiedantibodies, for example, wherein each functional equivalent is definedby the ability to bind to CXCR5, inhibiting CXCR5 signaling ability orfunction, or inhibiting binding of CXCL13 and other ligands to CXCR5.The skilled artisan will understand that there is an overlap in thegroup of molecules termed “antibody fragments” and the group termed“functional equivalents.” Methods of producing functional equivalentswhich retain CXCR5 binding ability are known to the person skilled inthe art and are disclosed, for example, in WO 93/21319, EPO Ser. No.239,400, WO 89/09622, EPO Ser. No. 338,745 and EPO Ser. No. 332,424.

The functional equivalents of the present application also includemodified antibodies, e.g., antibodies modified by the covalentattachment of any type of molecule to the antibody. For example,modified antibodies include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, deamidation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand, linkage to a toxinor cytotoxic moiety or other protein etc. The covalent attachment neednot yield an antibody that is immune from generating an anti-idiotypicresponse. The modifications may be achieved by known techniques,including, but not limited to, specific chemical cleavage, acetylation,formylation, metabolic synthesis etc. Additionally, the modifiedantibodies may contain one or more non-classical amino acids.

Many techniques are available to one of ordinary skill in the art whichpermit the optimization of binding affinity. Typically, the techniquesinvolve substitution of various amino acid residues at the site ofinterest, followed by a screening analysis of binding affinity of themutant polypeptide for the cognate antigen or epitope.

Once the antibody is identified and isolated, it is often useful togenerate a variant antibody or mutant, or mutein, wherein one or moreamino acid residues are altered, for example, in one or more of thehypervariable regions of the antibody. Alternatively, or in addition,one or more alterations (e.g., substitutions) of framework residues maybe introduced in the antibody where these result in an improvement inthe binding affinity of the antibody mutant for CXCR5. Examples offramework region residues that can be modified include those whichnon-covalently bind antigen directly (Amit et al., Science 233:747-753(1986)); interact with/affect the conformation of a CDR (Chothia et al.,J Mol Biol 196:901-917 (1987)); and/or participate in the V_(L)-V_(H)interface (EP 239 400). In certain embodiments, modification of one ormore of such framework region residues results in an enhancement of thebinding affinity of the antibody for the cognate antigen. For example,from about one to about five framework residues may be altered in thisembodiment of the invention. Sometimes, this may be sufficient to yieldan antibody mutant suitable for use in preclinical trials, even wherenone of the hypervariable region residues have been altered. Normally,however, the antibody mutant can comprise one or more hypervariableregion alteration(s). The constant regions also can be altered to obtaindesirable or more desirable effector properties.

The hypervariable region residues which are altered may be changedrandomly, especially where the starting binding affinity of the parentantibody is such that randomly-produced antibody mutants can be readilyscreened for altered binding in an assay as taught herein.

One procedure for obtaining antibody mutants, such as CDR mutants, is“alanine scanning mutagenesis” (Cunningham & Wells, Science244:1081-1085 (1989); and Cunningham & Wells, Proc Nat Acad Sci USA84:6434-6437 (1991)). One or more of the hypervariable region residue(s)are replaced by alanine or polyalanine residue(s). Those hypervariableregion residue(s) demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other mutationsat or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. Similarsubstitutions can be attempted with other amino acids, depending on thedesired property of the scanned residues.

A more systematic method for identifying amino acid residues to modifycomprises identifying hypervariable region residues involved in bindingCXCR5 and those hypervariable region residues with little or noinvolvement with CXCR5 binding. An alanine scan of the non-bindinghypervariable region residues is performed, with each ala mutant testedfor enhancing binding to CXCR5. In another embodiment, those residue(s)significantly involved in binding CXCR5 are selected to be modified.Modification can involve deletion of a residue or insertion of one ormore residues adjacent to a residue of interest. However, normally themodification involves substitution of the residue by another amino acid.A conservative substitution can be a first substitution. If such asubstitution results in a change in biological activity (e.g., bindingaffinity), then another conservative substitution can be made todetermine if more substantial changes are obtained.

Even more substantial modification in an antibody range and presentationof biological properties can be accomplished by selecting an amino acidthat differs more substantially in properties from that normallyresident at a site. Thus, such a substitution can be made whilemaintaining: (a) the structure of the polypeptide backbone in the areaof the substitution, for example, as a sheet or helical conformation;(b) the charge or hydrophobicity of the molecule at the target site, or(c) the bulk of the side chain.

For example, the naturally occurring amino acids can be divided intogroups based on common side chain properties:

(1) hydrophobic: methionine (M or met), alanine (A or ala), valine (V orval), leucine (L or leu) and isoleucine (I or ile);

(2) neutral, hydrophilic: cysteine (C or cys), serine (S or ser),threonine (T or thr), asparagine (N or asn) and glutamine (Q or gln);

(3) acidic: aspartic acid (D or asp) and glutamic acid (E or glu);

(4) basic: histidine (H or his), lysine (K or lys) and arginine (R orarg);

(5) residues that influence chain orientation: glycine (G or gly) andproline (P or pro), and

(6) aromatic: tryptophan (W or trp), tyrosine (Y or tyr) andphenylalanine (F or phe).

Non-conservative substitutions can entail exchanging an amino acid withan amino acid from an other group. Conservative substitutions can entailexchange of one amino acid for another within a group.

Preferred amino acid substitutions include those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity and (4) confer or modify otherphysico-chemical or functional properties of such analogs. Analogs caninclude various muteins of a sequence other than the naturally occurringpeptide sequence. For example, single or multiple amino acidsubstitutions (preferably conservative amino acid substitutions) may bemade in the naturally-occurring sequence (preferably in the portion ofthe polypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence) unless of a change in the bulk orconformation of the R group or side chain, Proteins, Structures andMolecular Principles (Creighton, ed., W.H. Freeman and Company, New York(1984)); Introduction to Protein Structure (Branden & Tooze, eds.,Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature354:105 (1991).

Ordinarily, the antibody mutant with improved biological properties willhave an amino acid sequence having at least 75% amino acid sequenceidentity or similarity with the amino acid sequence of either the heavyor light chain variable domain of the parent anti-human CXCR5 antibody,at least 80%, at least 85%, at least 90% and often at least 95%identity. Identity or similarity with respect to parent antibodysequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e., same residue) orsimilar (i.e., amino acid residue from the same group based on commonside-chain properties, supra) with the parent antibody residues, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity.

Alternatively, antibody mutants can be generated by systematic mutationof the FR and CDR regions of the heavy and light chains, or the F_(c)region of the anti-CXCR5 antibody. Another procedure for generatingantibody mutants involves the use of affinity maturation using phagedisplay (Hawkins et al., J Mol Biol 254:889-896 (1992) and Lowman etal., Biochemistry 30(45):10832-10838 (1991)). Bacteriophage coat-proteinfusions (Smith, Science 228:1315 (1985); Scott & Smith, Science 249:386(1990); Cwirla et al. Proc Natl Acad Sci USA 8:309 (1990); Devlin et al.Science 249:404 (1990); Wells & Lowman, Curr Opin Struct Biol 2:597(1992); and U.S. Pat. No. 5,223,409) are known to be useful for linkingthe phenotype of displayed proteins or peptides to the genotype ofbacteriophage particles which encode them. The F_(ab) domains ofantibodies have also been displayed on phage (McCafferty et al., Nature348: 552 (1990); Barbas et al. Proc Natl Acad Sci USA 88:7978 (1991);and Garrard et al. Biotechnol 9:1373 (1991)).

Monovalent phage display consists of displaying a set of proteinvariants as fusions of a bacteriophage coat protein on phage particles(Bass et al., Proteins 8:309 (1990). Affinity maturation, or improvementof equilibrium binding affinities of various proteins, has previouslybeen achieved through successive application of mutagenesis, monovalentphage display and functional analysis (Lowman & Wells, J Mol Biol234:564 578 (1993); and U.S. Pat. No. 5,534,617), for example, byfocusing on the CDR regions of antibodies (Barbas et al., Proc Natl AcadSci USA 91:3809 (1994); and Yang et al., J Mol Biol 254:392 (1995)).

Libraries of many (for example, 10⁶ or more) protein variants, differingat defined positions in the sequence, can be constructed onbacteriophage particles, each of which contains DNA encoding theparticular protein variant. After cycles of affinity purification, usingan immobilized antigen, individual bacteriophage clones are isolated,and the amino acid sequence of the displayed protein is deduced from theDNA.

Following production of the antibody mutant, the biological activity ofthat molecule relative to the parent antibody can be determined astaught herein. As noted above, that may involve determining the bindingaffinity and/or other biological activities or physical properties ofthe antibody. In a preferred embodiment of the invention, a panel ofantibody mutants are prepared and are screened for binding affinity forthe antigen. One or more of the antibody mutants selected from thescreen are optionally subjected to one or more further biologicalactivity assays to confirm that the antibody mutant(s) have new orimproved properties. In preferred embodiments, the antibody mutantretains the ability to bind CXCR5 with a binding affinity similar to orbetter/higher than that of the parent antibody.

The antibody mutant(s) so selected may be subjected to furthermodifications, often depending on the intended use of the antibody. Suchmodifications may involve further alteration of the amino acid sequence,fusion to heterologous polypeptide(s) and/or covalent modifications. Forexample, a cysteine residue not involved in maintaining the properconformation of the antibody mutant may be substituted, generally withserine, to improve the oxidative stability of the molecule and toprevent aberrant cross-linking. Conversely, a cysteine may be added tothe antibody to improve stability (particularly where the antibody is anantibody fragment such as an F_(v) fragment).

Another type of antibody mutant has an altered glycosylation pattern.That may be achieved by deleting one or more carbohydrate moieties foundin the antibody and/or by adding one or more glycosylation sites thatare not present in the antibody. Glycosylation of antibodies istypically either N-linked to Asn or O-linked to Ser or Thr. Thetripeptide sequences, asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are common recognitionsequences for enzymatic attachment of a carbohydrate moiety to theasparagine side chain. N-acetylgalactosamine, galactose, fucose orxylose, for example, are bonded to a hydroxyamino acid, most commonlyserine or threonine, although 5-hydroxyproline or 5-hydroxylysine alsomay be used. Addition or substitution of one or more serine or threonineresidues to the sequence of the original antibody can enhance thelikelihood of O-linked glycosylation.

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance the effectiveness of theantibody. For example, cysteine residue(s) may be introduced in theF_(c) region, thereby allowing interchain disulfide bond formation inthat region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC), see Caronet al., J Exp Med 176:1191-1195 (1992) and Shopes, Immunol 148:2918-2922(1993). Alternatively, an antibody can be engineered which has dualF_(c) regions and may thereby have enhanced complement lysis and ADCCcapabilities, see Stevenson et al., Anti-Cancer Drug Design 3: 219 230(1989).

Covalent modifications of the antibody are included within the scope ofthe invention. Such may be made by chemical synthesis or by enzymatic orchemical cleavage of the antibody, if applicable. Other types ofcovalent modifications of the antibody are introduced into the moleculeby reacting targeted amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor with the N-terminal or C-terminal residue.

Cysteinyl residues can be reacted with α-haloacetates (and correspondingamines), such as chloroacetic acid or chloroacetamide, to yieldcarboxylmethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso can be derivatized by reactionwith bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercura-4-nitrophenol orchloro-7-nitrobenzo-2-oxa-1,3-diazole, for example.

Histidyl residues can be derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0. p-bromophenacyl bromide also can beused, the reaction is preferably performed in 0.1 M sodium cacodylate atpH 6.0.

Lysinyl and α terminal residues can be reacted with succinic or othercarboxylic acid anhydrides to reverse the charge of the residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters, such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea and 2,4-pentanedione, and the amino acid can betransaminase-catalyzed with glyoxylate.

Arginyl residues can be modified by reaction with one or severalconventional reagents, such as phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione and ninhydrin. Derivatization of arginine residuesoften requires alkaline reaction conditions. Furthermore, the reagentsmay react with lysine as well as the arginine ε-amino group.

The specific modification of tyrosyl residues can be made with aromaticdiazonium compounds or tetranitromethane. For example, N-acetylimidizoleand tetranitromethane are used to form O-acetyl tyrosyl species and3-nitro derivatives, respectively. Tyrosyl residues can be iodinatedusing ¹²⁵I or ¹³¹I to prepare labeled proteins for use in aradioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) can be modified by reactionwith carbodiimides (R—N═C═C—R′), where R and R′ can be different alkylgroups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues can be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively, underneutral or basic conditions. The deamidated form of those residues fallswithin the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of serinyl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (Creighton, Proteins: Structure and Molecular Properties,W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of theN-terminal amine and amidation of any C-terminal carboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. Those procedures donot require production of the antibody in a host cell that hasglycosylation capabilities for N-linked or O-linked glycosylation.Depending on the coupling mode used, the sugar(s) may be attached to:(a) arginine and histidine; (b) free carboxyl groups; (c) freesulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups,such as those of serine, threonine or hydroxyproline; (e) aromaticresidues such as those of phenylalanine, tyrosine or tryptophan; or (f)the amide group of glutamine. Such methods are described in WO 87/05330and in Aplin & Wriston, CRC Crit. Rev Biochem, pp. 259-306 (1981).

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylation, forexample, can require exposure of the antibody to the compound,trifluoromethanesulfonic acid, or an equivalent compound, resulting incleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described, for example, inHakimuddin et al. Arch Biochem Biophys 259:52 (1987) and in Edge et al.,Anal Biochem 118:131 (1981). Enzymatic cleavage of carbohydrate moietieson antibodies can be achieved by any of a variety of endoglycosidasesand exoglycosidases as described, for example, in Thotakura et al., MethEnzymol 138:350 (1987).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol or polyoxylalkylenes, in themanner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

Another technique preferred for obtaining mutants or muteins is affinitymaturation by phage display (Hawkins et al., J Mol Biol 254:889-896(1992); and Lowman et al., Biochemistry 30(45):10832-10838 (1991)).Briefly, several hypervariable region sites (e.g., 6-7 sites) aremutated to generate all possible amino acid substitutions at each site.The antibody mutants thus generated are displayed in monovalent fashionon phage particles as fusions to a protein found on the particles. Thephage expressing the various mutants can be cycled through rounds ofbinding selection, followed by isolation and sequencing of those mutantswhich display high affinity.

The method of selecting novel binding polypeptides can utilize a libraryof structurally related polypeptides. The library of structurallyrelated polypeptides, for example, fused to a phage coat protein, isproduced by mutagenesis, and is displayed on the surface of theparticle. The particles then are contacted with a target molecule andthose particles having the highest affinity for the target are separatedfrom those of lower affinity. The high affinity binders then areamplified by infection of a suitable bacterial host and the competitivebinding step is repeated. The process is repeated until polypeptides ofthe desired affinity are obtained.

Alternatively, multivalent phage (McCafferty et al. (1990) Nature348:552-554; and Clackson et al. (1991) Nature 352:624-628) also can beused to express random point mutations (for example, generated by use ofan error-prone DNA polymerase) to generate a library of phage antibodyfragments which then could be screened for affinity to CXCR5, Hawkins etal., (1992) J Mol Biol 254:889-896.

Preferably, during the affinity maturation process, the replicableexpression vector is under tight control of a transcription regulatoryelement, and the culturing conditions are adjusted so the amount ornumber of particles displaying more than one copy of the fusion proteinis less than about 1%. Also preferably, the amount of particlesdisplaying more than one copy of the fusion protein is less than 10% ofthe amount of particles displaying a single copy of the fusion protein.Preferably the amount is less than 20%.

Functional equivalents may be produced by interchanging different CDRsof different antibody chains within a framework or a composite FRderived from plural antibodies. Thus, for example, different classes ofantibody are possible for a given set of CDRs by substitution ofdifferent heavy chains, for example, IgG₁₋₄, IgM, IgA₁₋₂ or IgD, toyield differing CXCR5 antibody types and isotypes. Similarly, artificialantibodies within the scope of the invention may be produced byembedding a given set of CDRs within an entirely synthetic framework.

For example, a suitable framework and F_(c) portion to carry thevariable region or one or more CDR's of interest is obtained from anIgG4 molecule, which has reduced effector function.

In other embodiments, to enhance the properties of a CXCR5-bindingmolecule of interest, certain modifications can be made to the frameworkportion and/or F_(c) portion of the molecule carrying theantigen-binding portion of a molecule of interest. For example, aminoacid substitutions can be made to enhance or to reduce properties ofinterest. Thus, in an IgG4 molecule, substitutions at sites known toimpact function, for example, in the hinge region, in a region thatimpacts an effector function or that impacts F_(c) binding, for example,are suitable for modification. In an IgG4 molecule, substitutions, usingKabat numbering, at amino acid 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,247, 248, 249, 250, 251, 252, 253, 254, 255 and so on, or combinationsthereof, can be made to obtain desired properties. For example,substituting a proline for serine 241 can stabilize the tertiary andquaternary structures of the molecule (Mol Imm 30(1)105-108, 1993), andsubstituting glutamic acid for leucine 248 can dampen effectorfunction(s) (J Imm 164(4)1925-1033, 2000; and Clin Imm 98(2)164-174,2001).

Another beneficial property is obtaining an antibody derivative whichbinds CXCR5 but, for example, does not deplete B cells. That can beadvantageous as antibody production in a patient is not compromised.Treatment with such a reagent also facilitates a combination regimenwith a second drug for a particular indication that acts at a levelother than at the B cell. That may be at the level of the T cell, forexample.

Hence, for example, 16D7-HC1-LC3 was treated to contain twosubstitutions, S241P and L248E. The proline and glutamic acid residuesconfer desired properties on a CXCR5-binding molecule of interestcarrying an IgG4 framework, such as, stability and reduced effectorfunction.

The antibody fragments and functional equivalents of the presentinvention encompass those molecules with a detectable degree of specificbinding to CXCR5. A detectable degree of binding includes all values inthe range of at least 10-100%, preferably at least 50%, 60% or 70%, morepreferably at least 75%, 80%, 85%, 90%, 95% or 99% of the bindingability of an antibody of interest. Also included are equivalents withan affinity greater than 100% that of an antibody of interest.

The CDRs generally are of importance for epitope recognition andantibody binding. However, changes may be made to residues that comprisethe CDRs without interfering with the ability of the antibody torecognize and to bind the cognate epitope. For example, changes that donot impact epitope recognition, yet increase the binding affinity of theantibody for the epitope, may be made. Several studies have surveyed theeffects of introducing one or more amino acid changes at variouspositions in the sequence of an antibody, based on the knowledge of theprimary antibody sequence, on the properties thereof, such as bindingand level of expression (Yang et al., 1995, J Mol Biol 254:392-403;Rader et al., 1998, Proc Natl Acad Sci USA 95:8910-8915; and Vaughan etal., 1998, Nature Biotechnology 16, 535-539).

Thus, equivalents of an antibody of interest can be generated bychanging the sequences of the heavy and light chain genes in the CDR1,CDR2 or CDR3, or framework regions, using methods such asoligonucleotide-mediated site-directed mutagenesis, cassettemutagenesis, error-prone PCR, DNA shuffling or mutator-strains of E.coli (Vaughan et al., 1998, Nat Biotech 16:535-539; and Adey et al.,1996, Chap. 16, pp. 277-291, in Phage Display of Peptides and Proteins,eds. Kay et al., Academic Press). The methods of changing the nucleicacid sequence of the primary antibody can result in antibodies withimproved affinity (Gram et al., 1992, Proc Natl Acad Sci USA89:3576-3580; Boder et al., 2000, Proc Natl Acad Sci USA 97:10701-10705;Davies & Riechmann, 1996, Immunotech 2:169-179; Thompson et al., 1996, JMol Biol 256:77-88; Short et al., 2002, J Biol Chem 277:16365-16370; andFurukawa et al., 2001, J Biol Chem 276:27622-27628).

Repeated cycles of “polypeptide selection” can be used to select forhigher and higher affinity binding by, for example, the selection ofmultiple amino acid changes which are selected by multiple selection ofcycles. Following a first round of selection, involving a first regionof selection of amino acids in the ligand or antibody polypeptide,additional rounds of selection in other regions or amino acids of theligand are conducted. The cycles of selection are repeated until thedesired affinity properties are achieved.

Improved antibodies also include those antibodies having improvedcharacteristics that are prepared by the standard techniques of animalimmunization, hybridoma formation and selection for antibodies withspecific characteristics.

“Antagonist” refers to a molecule capable of inhibiting one or morebiological activities of a target molecule, such as signaling by CXCR5.Antagonists may interfere with the binding of a receptor to a ligand andvice versa, by incapacitating or killing cells activated by a ligand,and/or by interfering with receptor or ligand activation (e.g., tyrosinekinase activation) or signal transduction after ligand binding to areceptor. The antagonist may completely block receptor-ligandinteractions or may substantially reduce such interactions. All suchpoints of intervention by an antagonist shall be considered equivalentfor purposes of the instant invention. Thus, included within the scopeof the invention are antagonists (e.g., neutralizing antibodies) thatbind to CXCR5, CXCL13 or other ligands of CXCR5, or a complex of CXCR5and a ligand thereof, such as CXCL13; amino acid sequence variants orderivatives of CXCR5 or CXCL13 which antagonize the interaction betweenCXCR5 and a ligand, such as CXCL13; soluble CXCR5, optionally fused to aheterologous molecule such as an immunoglobulin region (e.g., animmunoadhesin); a complex comprising CXCR5 in association with anotherreceptor or biological molecule; synthetic or native sequence peptideswhich bind to CXCR5; and so on.

“Agonist” refers to a compound, including a protein, a polypeptide, apeptide, an antibody, an antibody fragment, a conjugate, a largemolecule, a small molecule, which activates one or more biologicalactivities of CXCR5. Agonists may interact with the binding of areceptor to a ligand and vice versa, by acting as a mitogen of cellsactivated by a ligand, and/or by interfering with cell inactivation orsignal transduction inhibition after ligand binding to a receptor. Allsuch points of intervention by an agonist shall be considered equivalentfor purposes of the instant invention. Thus, included within the scopeof the invention are agonists that bind to CXCR5, CXCL13 or other ligandof CXCR5, or a complex of CXCR5 and a ligand thereof, such as CXCL13;amino acid sequence variants or derivatives of CXCR5 or CXCL13 whichfacilitate the interaction between CXCR5 and a ligand, such as CXCL13;soluble CXCR5, optionally fused to a heterologous molecule such as animmunoglobulin region (e.g., an immunoadhesin); a complex comprisingCXCR5 in association with another receptor or biological molecule;synthetic or native sequence peptides which bind to CXCR5; and so on.The agonist generally is an entity which directly activates CXCR5, forexample, to signal.

The terms “cell,” “cell line,” and “cell culture” include progenythereof. It is also understood that all progeny may not be preciselyidentical, such as in DNA content, due to deliberate or inadvertentmutation. Variant progeny that have the same function or biologicalproperty of interest, as screened for in the original cell, areincluded. The “host cells” used in the present invention generally areprokaryotic or cukaryotic hosts, selected as a design choice.

“Transformation” of a cellular organism, cell or cell line with anucleic acid means introducing a nucleic acid into the target cell sothat the nucleic acid is replicable, either as an extrachromosomalelement or by chromosomal integration, and, optionally, expressed.“Transfection” of a cell or organism with a nucleic acid refers to thetaking up of the nucleic acid, e.g., an expression vector, by the cellor organism whether or not any coding sequences are in fact expressed.The terms “transfected host cell” and “transformed” refer to a cell inwhich a nucleic acid was introduced. Typical prokaryotic host cellsinclude various strains of E. coli. Typical eukaryotic host cells aremammal cells, such as Chinese hamster ovary, or cells of human origin.The introduced nucleic acid sequence may be from the same species as thehost cell or of a different species from the host cell, or may be ahybrid nucleic acid sequence, containing some foreign and somehomologous nucleic acids. Transformation can also occur by transductionor infection with virus-derived elements.

The term “vector” means a nucleic acid construct, a carrier, containinga nucleic acid, the transgene, the foreign gene or the gene of interest,which can be operably linked to suitable control sequences forexpression of the transgene in a suitable host. Such control sequencesinclude, for example, a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encodingsuitable mRNA ribosome binding sites and sequences which control thetermination of transcription and translation. The vector may be aplasmid, a phage particle or just a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may in some instances, integrateinto the host cell genome. In the present specification, “plasmid” and“vector” are used interchangeably, as the plasmid is a commonly usedform of vector. However, the invention is intended to include such otherforms of vectors which serve equivalent carrier function as and whichare, or become, known in the art, such as viruses, synthetics moleculesthat carry nucleic acids, liposomes and the like.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including human, domestic and farm animals, nonhuman primates,and zoo, sports or pet animals, such as dogs, horses, cats, cows etc.

The antibodies of interest can be screened or can be used in an assay asdescribed herein or as known in the art. Often, such assays require areagent to be detectable, that is, for example, labeled. The word“label” when used herein refers to a detectable compound or compositionwhich can be conjugated directly or indirectly to a molecule or protein,e.g., an antibody. The label may itself be detectable (e.g.,radioisotope labels, particles or fluorescent labels) or, in the case ofan enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

As used herein, “solid phase” means a non-aqueous matrix to which anentity or molecule, such as the antibody of the instant invention, canadhere. Example of solid phases encompassed herein include those formedpartially or entirely of glass (e.g., controlled pore glass),polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinylalcohol and silicones. In certain embodiments, depending on the context,the solid phase can comprise the well of an assay plate; in others canbe used in a purification column (e.g., an affinity chromatographycolumn). Thus, the solid phase can be a paper, a bead, a plastic, a chipand so on, can be made from a variety of materials, such asnitrocellulose, agarose, polystyrene, polypropylene, silicon and so on,and can be in a variety of configurations.

Soluble CXCR5 or fragments thereof, such as the extracellular domain(EC) domain, can be used as immunogens for generating antibodies ofinterest. The immunogen can be obtained or isolated from natural sourcesor can be made recombinantly. Whole cells, such as CXCR5⁺ cells, cellsderived from a natural source (e.g., B cell, B cell lines or cancer celllines) or cells transformed (or transfected) by recombinant techniquesto express, and perhaps to overexpress CXCR5, may be used as theimmunogen for making the antibodies of interest. Also, membranepreparations carrying CXCR5 or synthetic peptides or truncatedpolypeptides corresponding to the EC regions of CXCR5 can be used, asknown in the art.

The EC, which is about 60 amino acids in length, or portions thereof, ofCXCR5 can be used as the immunogen. Other forms of the immunogen usefulfor preparing antibodies, such as a conjugate, will be apparent to thosein the art. Thus, CXCR5, or portions thereof, can be attached to acarrier molecule, such as albumin or KLH, to be used as an immunogen. Ofcourse, with cells expressing CXCR5, it is the EC domain which is thepreferred immunogen or portion of the immunogen.

The gene or a cDNA encoding CXCR5, as known in the art, may be cloned ina plasmid or other expression vector and expressed in any of a number ofexpression systems according to methods well known to those of skill inthe art, and see below, for example. Because of the degeneracy of thegenetic code, a multitude of nucleotide sequences encoding CXCR5 proteinor polypeptides may be used in the practice of expressing recombinantCXCR5 or functional products thereof. The nucleotide sequence may varyby selecting combinations based on possible codon choices, such as thosepreferred by the host cell. The combinations are made in accordance withthe standard triplet genetic code as applied to the nucleotide sequencethat codes for naturally occurring CXCR5 and all such variations may beconsidered. Thus, the CXCR5-encoding sequence can be recoded to containcodons expressing the amino acid of interest, however, the triplet codonis one favored by the gene expression machinery of the host cell, suchas a human cell. Any one of those polypeptides may be used in theimmunization of an animal, such as a camelid, or other system togenerate antibodies that bind to CXCR5.

As mentioned above, the CXCR5 immunogen may, when beneficial, beexpressed as a fusion protein that has CXCR5 attached to a fusionsegment, which generally is a polypeptide with one or more beneficialfunctions. The fusion segment often aids in protein purification, e.g.,by permitting the fusion protein to be isolated and purified by affinitychromatography, but can also be used to increase immunogenicity. Fusionproteins can be produced by culturing a recombinant cell transformedwith a fusion nucleic acid sequence that encodes a protein attached toeither the carboxyl and/or amino terminal end of the CXCR5 polypeptide.Fusion segments may include, but are not limited to, immunoglobulinF_(c) regions, glutathione-S-transferase, β-galactosidase, apoly-histidine segment capable of binding to a divalent metal ion andmaltose binding protein.

Nucleic acid molecules encoding amino acid sequence mutants can beprepared by a variety of methods known in the art. The methods include,but are not limited to, oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis and cassette mutagenesis of an earlierprepared mutant or a non-mutant version of the molecule of interest,(see, for example, Kunkel, Proc Natl Acad Sci USA 82:488 (1985)).

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention, a single chain antibody of the invention oran antibody mutein of the invention) includes construction of anexpression vector containing a polynucleotide that encodes the antibodyor a fragment of the antibody as described herein. Once a polynucleotideencoding an antibody molecule has been obtained, the vector for theproduction of the antibody may be produced by recombinant DNA technologyas known in the art. An expression vector is constructed containingantibody coding sequences and appropriate transcriptional andtranslational control signals. The methods include, for example, invitro recombinant DNA techniques, synthetic techniques and in vivogenetic recombination.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells then are cultured by conventionaltechniques to produce an antibody or fragment of the invention. In oneaspect of the invention, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed herein.

A variety of host/expression vector systems may be utilized to expressthe antibody molecules of the invention. Such expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ.Bacterial cells, such as E. coli, and eukaryotic cells are commonly usedfor the expression of a recombinant antibody molecule, especially forthe expression of whole recombinant antibody molecule. For example,mammal cells such as CHO cells, in conjunction with a vector, such asone carrying the major intermediate early gene promoter element fromhuman cytomegalovirus, are an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); and Cockett et al., Bio/Technology8:2 (1990)). Plants and plant cell culture, insect cells and so on alsocan be used to make the proteins of interest, as known in the art.

In addition, a host cell is chosen which modulates the expression of theinserted sequences, or modifies and processes the gene product in thespecific fashion desired. Such modifications (e.g., glycosylation) andprocessing (e.g., cleavage) of protein products may be important for thefunction of the protein. Different host cells have characteristic andspecific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification andprocessing of the expressed antibody of interest. Hence, eukaryotic hostcells which possess the cellular machinery for proper processing of theprimary transcript, glycosylation and phosphorylation of the geneproduct may be used. Such mammalian host cells include, but are notlimited to, CHO, COS, 293, 3T3 or myeloma cells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites etc.) and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for one to two days in an enriched media, and then aremoved to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into a chromosome and be expanded into a cellline. Such engineered cell lines not only are useful for antibodyproduction but are useful in screening and evaluation of compounds thatinteract directly or indirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe Herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska etal., Proc Natl Acad Sci USA 48:202 (1992)), glutamate synthase selectionin the presence of methionine sulfoximide (Adv Drug Del Rev 58, 671,2006 and see the website or literature of Lonza Group Ltd.) and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes in tk,hgprt or aprt cells, respectively. Also, antimetabolite resistance canbe used as the basis of selection for the following genes: dhfr, whichconfers resistance to methotrexate (Wigler et al., Proc Natl Acad SciUSA 77:357 (1980); O'Hare et al., Proc Natl Acad Sci USA 78:1527(1981)); gpt, which confers resistance to mycophenolic acid (Mulligan etal., Proc Natl Acad Sci USA 78:2072 (1981)); neo, which confersresistance to the aminoglycoside, G-418 (Wu et al., Biotherapy 3:87(1991)); and hygro, which confers resistance to hygromycin (Santerre etal., Gene 30:147 (1984)). Methods known in the art of recombinant DNAtechnology may be routinely applied to select the desired recombinantclone, and such methods are described, for example, in Ausubel et al.,eds., Current Protocols in Molecular Biology, John Wiley & Sons (1993);Kriegler, Gene Transfer and Expression, A Laboratory Manual, StocktonPress (1990); Dracopoli et al., eds., Current Protocols in HumanGenetics, John Wiley & Sons (1994); and Colberre-Garapin et al., J MolBiol 150:1 (1981).

The expression levels of an antibody molecule can be increased by vectoramplification (for example, see Bebbington et al., in DNA Cloning, Vol.3. Academic Press (1987)). When a marker in the vector system expressingantibody is amplifiable, an increase in the level of inhibitor presentin the culture will increase the number of copies of the marker gene.Since the amplified region is associated with the antibody gene,production of the antibody will also increase (Crouse et al., Mol CellBiol 3:257 (1983)).

The host cell may be co-transfected with two or more expression vectorsof the invention, for example, the first vector encoding a heavychain-derived polypeptide and the second vector encoding a lightchain-derived polypeptide. The two vectors may contain identicalselectable markers which enable equal expression of heavy and lightchain polypeptides. Alternatively, a single vector may be used whichencodes, and is capable of expressing, both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, Nature 322:52 (1986); and Kohler, Proc Natl Acad Sci USA77:2197 (1980)). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for CXCR5 after Protein Aand size-exclusion chromatography and so on), centrifugation,differential solubility or by any other standard technique for thepurification of proteins. In addition, the antibodies of the instantinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

Recombinant CXCR5 protein, as exemplified in the examples below, wasused to immunize mice to generate the hybridomas that produce monoclonalantibodies of the present invention. The monoclonals obtained wereselected for those with beneficial therapeutic potential, for example,preventing binding of CXCR5 ligand thereto. The selected antibodies thenwere modified to obtain beneficial properties, such as having enhancedstability in vivo.

The antibodies of the present invention may be generated by any suitablemethod known in the art. The antibodies of the present invention maycomprise polyclonal antibodies, although because of the modification ofantibodies to optimize use in human, as well as to optimize the use ofthe antibody per se, monoclonal antibodies are preferred because of easeof production and manipulation of particular proteins. Methods ofpreparing polyclonal antibodies are known to the skilled artisan (Harlowet al., Antibodies: a Laboratory Manual, Cold Spring Harbor LaboratoryPress, 2nd ed. (1988)).

For example, an immunogen, as exemplified herein, may be administered tovarious host animals including, but not limited to, rabbits, mice,camelids, rats etc., to induce the production of serum containingpolyclonal antibodies specific for CXCR5. The administration of theimmunogen may entail one or more injections of an immunizing agent and,if desired, an adjuvant. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral oil,gels, alum (aluminum hydroxide), surface active substances, such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins (KLH), dinitrophenol and potentially usefulhuman adjuvants, such as BCG (Bacille Calmette-Guerin) andCorynebacterium parvum. Additional examples of adjuvants which may beemployed include the MPL-TDM adjuvant (monophosphoryl lipid A, synthetictrehalose dicorynomycolate). Immunization protocols are well known inthe art and may be performed by any method that elicit an immuneresponse in the animal host chosen. Thus, various administration routescan be used over various time periods as a design choice.

Typically, the immunogen (with or without adjuvant) is injected into themammal by multiple subcutaneous or intraperitoneal injections, orintramuscularly or intravenously. The immunogen may include a CXCR5polypeptide, a fusion protein, or variants thereof, which may beproduced by a cell that produces or overproduces CXCR5, which may be anaturally occurring cell, a naturally occurring mutant cell or agenetically engineered cell. In certain circumstances, whole cellsexpressing CXCR5 can be used. Depending on the nature of thepolypeptides (i.e., percent hydrophobicity, percent hydrophilicity,stability, net charge, isoelectric point etc.), the CXCR5 or portionthereof may be modified or conjugated to be immunogenic or moreimmunogenic in the animal, such as a mammal, being immunized. Forexample, CXCR5 or a portion thereof can be conjugated to a carrier. Theconjugation includes either chemical conjugation by derivatizing activechemical functional groups to both the immunogen and the immunogenicprotein to be conjugated such that a covalent bond is formed, or throughfusion-protein based methodology, or other methods known to the skilledartisan. Examples of such carriers or immunogenic proteins include, butare not limited to, KLH, ovalbumin, serum albumin, bovine thyroglobulin,soybean trypsin inhibitor, and promiscuous T helper peptides. Variousadjuvants may be used to increase the immunological response asdescribed above.

Once a suitable preparation is obtained, it is possible to isolateparticular antibodies from the plural antibodies by known separationtechniques, such as affinity chromatography, panning, absorption and soon. In that way, an individual antibody species can be obtained forfurther study, for example, sequencing to obtain the amino acidsequences of one or more CDRs.

The antibodies of the present invention preferably comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomatechnology, such as described by Kohler et al., Nature 256:495 (1975);U.S. Pat. No. 4,376,110; Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 2nd ed. (1988) and Hammerling etal., Monoclonal Antibodies and T-Cell Hybridomas, Elsevier (1981),recombinant DNA methods, for example, making and using transfectomas, orother methods known to the artisan. Other examples of methods which maybe employed for producing monoclonal antibodies include, but are notlimited to, the human B-cell hybridoma technique (Kosbor et al.,Immunology Today 4:72 (1983); and Cole et al., Proc Natl Acad Sci USA80:2026 (1983)), and the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss(1985)). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA and IgD, and any subclass thereof. The hybridomaproducing the mAb of the invention may be cultivated in vitro or invivo.

In the hybridoma model, a host such as a mouse, a humanized mouse, atransgenic mouse with human immune system genes, hamster, rabbit, rat,camel or any other appropriate host animal, is immunized to elicitlymphocytes that produce or are capable of producing antibodies thatspecifically bind to CXCR5. Alternatively, lymphocytes may be immunizedin vitro. Lymphocytes then are fused with myeloma cells using a suitablefusing agent, such as polyethylene glycol, to form a hybridoma cell(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,pp. 59-103 (1986)).

Generally, in making antibody-producing hybridomas, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine or human origin. Typically, a rat or mouse myeloma cell line isemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin and thymidine (“HATmedium”), substances that prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asthose derived from the MOPC-21 and MPC-11 mouse tumors available fromthe Salk Institute Cell Distribution Center, San Diego, Calif. andSP2/0, FO or X63-Ag8-653 cells available from the American Type CultureCollection, Manassas, Va.

Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, JImmunol 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, Marcel Dekker, Inc, pp. 51-63(1987)). The mouse myeloma cell line NSO may also be used (EuropeanCollection of Cell Cultures, Salisbury, Wilshire, UK).

Another alternative is to use electrical fusion rather than chemicalfusion to form hybridomas. Instead of fusion, a B cell can beimmortalized using, for example, Epstein Barr Virus or anothertransforming gene, see, e.g., Zurawaki et al., in Monoclonal Antibodies,ed., Kennett et al., Plenum Press, pp. 19-33. (1980). Transgenic miceexpressing immunoglobulins and severe combined immunodeficient (SCID)mice transplanted with human B lymphocytes also can be used.

The culture medium in which hybridoma cells are grown is assayed forproduction of monoclonal antibodies directed against CXCR5. The bindingspecificity of monoclonal antibodies produced by hybridoma cells may bedetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA), fluorocytometric analysis (FACS) orenzyme-linked immunosorbent assay (ELISA). Such techniques are known inthe art and are within the skill of the artisan. The binding affinity ofthe monoclonal antibody to CXCR5 can, for example, be determined by aScatchard analysis (Munson et al., Anal Biochem 107:220 (1980)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,pp. 59-103 (1986)). Suitable culture media include, for example,Dulbecco's Modified Eagle's Medium (D-MEM) or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated or isolated from the culture medium, ascites fluid or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharosc, protein G-Sepharosc, hydroxylapatitechromatography, gel exclusion chromatography, gel electrophoresis,dialysis or affinity chromatography.

A variety of methods exist in the art for the production of monoclonalantibodies and thus, the invention is not limited to their soleproduction in hybridomas. For example, the monoclonal antibodies may bemade by recombinant DNA methods, such as those described in U.S. Pat.No. 4,816,567. In this context, the term “monoclonal antibody” refers toan antibody derived from a single eukaryotic, phage or prokaryoticclone.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies, or such chainsfrom human, humanized or other sources) (Innis et al. in PCR Protocols.A Guide to Methods and Applications, Academic (1990), and Sanger et al.,Proc Natl Acad Sci 74:5463 (1977)). The hybridoma cells serve as asource of such DNA. Once isolated, the DNA may be placed into expressionvectors, which are then transfected into host cells such as E. colicells, NS0 cells, COS cells, Chinese hamster ovary (CHO) cells ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. The DNA also may be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences (U.S. Pat. No. 4,816,567; andMorrison et al., Proc Natl Acad Sci USA 81:6851 (1984)) or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneCXCR5-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the F_(c) region so as to prevent heavy chain cross-linking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to preventcross-linking.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,J Biochem Biophys Methods 24:107 (1992); and Brennan et al., Science229:81 (1985)). For example, F_(ab) and F_((ab′)2) fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce F_(ab) fragments) orpepsin (to produce F_((ab′)2) fragments). F_((ab′)2) fragments containthe variable region, the light chain constant region and the constantregion C_(H1) domain of the heavy chain. However, those fragments can beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from an antibody phage library. Alternatively,F_((ac′)2)-SH fragments can be directly recovered from E. coli andchemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163 (1992). According to another approach, F_((ab′)2)fragments can be isolated directly from recombinant host cell culture.Other techniques for the production of antibody fragments will beapparent to the skilled practitioner. In other embodiments, the antibodyof choice is a single chain F_(v) fragment (F_(v)) (WO 93/16185).

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanizedor human antibodies. Methods for producing chimeric antibodies are knownin the art, see e.g., Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986); Gillies et al., J Immunol Methods 125:191(1989); and U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397.

Humanized antibodies are derived from antibody molecules generated in anon-human species that bind CXCR5 wherein one or more CDRs therefrom areinserted into the FR regions from a human immunoglobulin molecule.Antibodies can be humanized using a variety of techniques known in theart including, for example, CDR grafting (EPO 239,400; WO 91/09967; andU.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering orresurfacing (EPO 592,106; EPO 519,596; Padlan, Molecular Immunology28:489 (1991); Studnicka et al., Protein Engineering 7:805 (1994); andRoguska et al., Proc Natl Acad Sci USA 91:969 (1994)), and chainshuffling (U.S. Pat. No. 5,565,332).

A humanized antibody has one or more amino acid residues from a sourcethat is non-human. The non-human amino acid residues are often referredto as “import” residues, which are typically taken from an “import”variable domain. Humanization can be essentially performed following themethods of Winter and co-workers (Jones et al., Nature 321:522 (1986);Ricchmann et al., Nature 332:323 (1988); and Verhoeyen et al., Science239:1534 (1988)), by substituting non-human CDRs or portions of CDRsequences for the corresponding sequences of a human antibody.Accordingly, such “humanized” antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possible some FRresidues are substituted from analogous sites in rodent antibodies. Theheavy chain constant region, which can include one or more heavy chaindomains, and hinge region can be from any class or subclass to obtain adesired effect, such as a particular effector function.

Often, framework residues in the human framework regions can besubstituted with the corresponding residue from the CDR donor antibodyto alter, and possibly improve, antigen binding. The frameworksubstitutions are identified by methods known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions, see, e.g., U.S. Pat. No. 5,585,089; and Riechmann et al.,Nature 332:323 (1988).

It is further preferable that humanized antibodies retain high affinityfor CXCR5, and retain or acquire other favorable biological properties.Thus, humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthe displays permits analysis of the likely role of certain residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind CXCR5. In that way, FR residues can be selectedand combined from the recipient and import sequences so that the desiredantibody characteristic, such as increased affinity for the targetantigen, is maximized, although it is the CDR residues that directly andmost substantially influence CXCR5 binding. The CDR regions also can bemodified to contain one or more amino acids that vary from that obtainedfrom the parent antibody from which the CDR was obtained, to provideenhanced or different properties of interest, such as binding of greateraffinity or greater avidity, for example.

Certain portions of the constant regions of antibody can be manipulatedand changed to provide antibody homologs, derivatives, fragments and thelike with properties different from or better than that observed in theparent antibody. Thus, for example, many IgG4 antibodies form intrachaindisulfide bonds near the hinge region. The intrachain bond candestabilize the parent bivalent molecule forming monovalent moleculescomprising a heavy chain with the associated light chain. Such moleculescan reassociate, but on a random basis.

It was observed that modifying amino acids in the hinge region of IgG4molecules can reduce the likelihood of intrachain bond formation,thereby stabilizing the IgG4 molecule, which will minimize thelikelihood of forming bispecific molecules. That modification can bebeneficial if a therapeutic antibody is an IgG4 molecule as the enhancedstability will minimize the likelihood of having the molecule dissociateduring production and manufacture, as well as in vivo. A monovalentantibody may not have the same effectiveness as the bivalent parentmolecule. For example, when bivalent IgG4 is administered to a patient,the percentage of bivalent IgG4 decays to about 30% over a two-weekperiod. An amino acid substitution at position 228 enhances IgG4stability. The serine that resides at 228 can be replaced with anotheramino acid, such as one of the remaining 19 amino acids. Such a changecan be made particularly with recombinant antibodies wherein the nucleicacid coding sequence can be mutated to yield a replacement amino acid atposition 228. For example, the S can be replaced with a proline.

Another set of amino acids suitable for modification include amino acidsin the area of the hinge which impact binding of a molecule containing aheavy chain with binding to the F_(c) receptor and internalization ofbound antibody. Such amino acids include, in IgG1 molecules, residuesfrom about 233 to about 237 (Glu-Leu-Leu-Gly-Gly); (SEQ ID NO:49) fromabout 252 to about 256 (Met-Ile-Ser-Arg-Thr) (SEQ ID NO:50) and fromabout 318 (Glu) to about 331 (Pro), including, for example, Lys₃₂₀,Lys₃₂₂ and Pro₃₂₉.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences, see, U.S. Pat. Nos. 4,444,887 and 4,716,111; and WO 98/46645,WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO91/10741. The techniques of Cole et al. and Boerder et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss (1985); andBoerner et al., J Immunol 147:86 (1991)).

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichalso express certain human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region and diversityregion may be introduced into mouse embryonic stem cells, in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of the human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies, see, e.g., Jakobovitis et al.,Proc Natl Acad Sci USA 90:2551 (1993); Jakobovitis et al., Nature362:255 (1993); Bruggermann et al., Year in Immunol 7:33 (1993); andDuchosal et al., Nature 355:258 (1992)).

The transgenic mice are immunized in the normal fashion with a CXCR5,e.g., all or a portion of CXCR5, such as the EC domain thereof.Monoclonal antibodies directed against CXCR5 can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview, see Lonberg et al., Int Rev Immunol13:65-93 (1995). For a discussion of producing human antibodies andhuman monoclonal antibodies and protocols for producing such antibodies,see, e.g., WO 98/24893; WO 92/01047; WO 96/34096; and WO 96/33735; EPONo. 0 598 877; and U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425;5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and5,939,598. In addition, companies such as Amgen (Fremont, Calif.),Genpharm (San Jose, Calif.) and Medarex, Inc. (Princeton, N.J.) can beengaged to provide human antibodies directed against CXCR5 usingtechnology similar to that described above.

Also, human mAbs could be made by immunizing mice transplanted withhuman peripheral blood leukocytes, splenocytes or bone marrow (e.g.,trioma technique of XTL Biopharmaceuticals, Israel). Completely humanantibodies which recognize a selected epitope can be generated using atechnique referred to as “guided selection.” In that approach, aselected non-human monoclonal antibody, e.g., a mouse antibody, is usedto guide the selection of a completely human antibody recognizing thesame epitope (Jespers et al., Bio/technology 12:899 (1988)).

When using recombinant techniques, the antibody variant can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody variant is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, maybe removed, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is exposed to sodium acetate (pH 3.5) and EDTA. Celldebris can be removed by centrifugation. Where the antibody variant issecreted into the medium, supernatant from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedto inhibit proteolysis, and antibiotics may be included to preventgrowth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis and affinity chromatography. The suitability of protein A orprotein G as an affinity ligand depends on the species and isotype ofany immunoglobulin F_(v) domain that is present in the antibody variant.Protein A can be used to purify antibodies that are based on human IgG1,IgG2 or IgG4 heavy chains (Lindmark et al., J Immunol Meth 62:1 (1983)).Protein G can be used for mouse isotypes and for human IgG3 (Cuss etal., EMBO J. 5:1567 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices, such as controlled pore glass orpoly(styrenedivinyl)benzene, allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodyvariant comprises a C_(H3) domain, the Bakerbond ABXTM resin (JT Baker;Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification, such as fractionation on an ion-exchange column,ethanol precipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin agarose chromatography on an anion or cationexchange resin (such as a polyaspartic acid column), chromatofocusing,SDS-PAGE and ammonium sulfate precipitation are also available,depending on the antibody or variant to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody or variant of interest and contaminants may be subjected tolow pH hydrophobic interaction chromatography using an elution buffer ata pH of between about 2.5-4.5, preferably performed at low saltconcentrations (e.g., from about 0-0.25 M salt).

Further, antibodies of the invention can, in turn, be utilized togenerate anti-idiotype antibodies that “mimic” CXCR5 using techniqueswell known to those skilled in the art (see, e.g., Greenspan et al.,FASEB J 7:437 (1989); and Nissinoff, J Immunol 147:2429 (1991)). Forexample, antibodies which bind to and competitively inhibitmultimerization and/or binding of a ligand to CXCR5 can be used togenerate anti-idiotypes that “mimic” CXCR5 and binding domain and, as aconsequence, bind to and neutralize CXCR5 and/or its ligand. Suchneutralizing anti-idiotypes or F_(ab) fragments of such anti-idiotypescan be used in therapeutic regimens, for example, to neutralize CXCL13.

The antibodies of the present invention may be bispecific antibodies.Bispecific antibodies can be monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present invention, one of the binding specificities isdirected towards CXCR5, the other may be for any other antigen, such asa cell-surface protein, receptor, receptor subunit, ligand,tissue-specific antigen, virally-derived protein, virally-encodedenvelope protein, bacterially-derived protein, bacterial surface proteinetc. Thus, the other specificity could be to CXCL13.

Methods for making bispecific antibodies are well known. Traditionally,the recombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy chain/light chain pairs, wherethe two heavy chains have different specificities (Milstein et al.,Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, the hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829 and in Trauneckeret al., EMBO J. 10:3655 (1991). Other methods for making bispecificantibodies are provided in, for example, Kufer et al., Trends Biotech22:238-244, 2004.

Antibody variable domains with the desired binding specificities can befused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H2), and C_(H3) regions. It may have thefirst heavy chain constant region (C_(H1)) containing the site necessaryfor light chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transformed into a suitable host organism. Forfurther details of generating bispecific antibodies see, for exampleSuresh et al., Meth Enzym 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980).It is contemplated that the antibodies may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioester bond. Examplesof suitable reagents for that purpose include iminothiolate andmethyl-4-mercaptobutyrimidate, and those disclosed, for example, in U.S.Pat. No. 4,676,980.

In addition, one can generate single-domain antibodies to CXCR5.Examples of that technology have been described in WO9425591 forantibodies derived from Camelidae heavy chain Ig, as well as inUS20030130496 describing the isolation of single domain fully humanantibodies from phage libraries.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423 (1988);Huston et al., Proc Natl Acad Sci USA 85:5879 (1988); and Ward, et al.,Nature 334:544 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the F_(v) region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional F_(v) fragments in E. coli may also be used (Skerra et al.,Science 242:1038 (1988)).

The instant invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide. Fused or conjugated antibodies of thepresent invention may be used for ease in purification, see e.g., WO93/21232; EP 439,095; Naramura et al., Immunol Lett 39:91 (1994); U.S.Pat. No. 5,474,981; Gillies et al., Proc Natl Acad Sci USA 89:1428(1992); and Fell et al., J Immunol 146:2446 (1991). The marker aminoacid sequence can be a hexa-histidine peptide (SEQ ID NO:51), such asthe tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, Calif.),among others, many of which are commercially available, Gentz et al.,Proc Natl Acad Sci USA 86:821 (1989). Other peptide tags useful forpurification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

One can also create a single peptide chain binding molecules in whichthe heavy and light chain F_(v) regions are connected. Single chainantibodies (“scF_(v)”) and the method of their construction aredescribed in, for example, U.S. Pat. No. 4,946,778. Alternatively,F_(ab) can be constructed and expressed by similar means. All of thewholly and partially human antibodies can be less immunogenic thanwholly murine mAbs, and the fragments and single chain antibodies alsocan be less immunogenic.

Antibodies or antibody fragments can be isolated from antibody phagelibraries generated using the techniques described in McCafferty et al.,Nature 348:552 (1990). Clarkson et al., Nature 352:624 (1991) and Markset al., J Mol Biol 222:581 (1991) describe the isolation of murine andhuman antibodies, respectively, using phage libraries. Subsequentpublications describe the production of high affinity (nM range) humanantibodies by chain shuffling (Marks et al., Bio/Technology 10:779(1992)), as well as combinatorial infection and in vivo recombination asa strategy for constructing very large phage libraries (Waterhouse etal., Nucl Acids Res 21:2265 (1993)). Thus, the techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

Anti-CXCR5 antibodies are tested by enzyme-linked immunosorbent assay(ELISA), FACS, Western immunoblotting or other immunochemical techniquesas known in the art. Thus, B cells or cells expressing CXCR5 can be usedto detect antibody binding thereto using a known technique, orrecombinantly expressed CXCR5 or portion thereof, such as the EC domain,can be adhered to a solid phase and used as a capture element in anassay, configured as a design choice.

To determine whether a particular antibody homolog binds to human CXCR5,any conventional binding assay may be used. Useful CXCR5 binding assaysinclude FACS analysis, ELISA assays, radioimmunoassays and the like,which detect binding of antibody, and functions resulting therefrom, tohuman CXCR5. Full-length and soluble forms of human CXCR5 taught hereinare useful in such assays. The binding of an antibody or homolog toCXCR5, or to soluble fragments thereof, may conveniently be detectedthrough the use of a second antibody specific for immunoglobulins of thespecies from which the antibody or homolog is derived.

To determine whether a particular antibody or homolog does or does notsignificantly block binding of CXCL13 or other ligand to human CXCR5,any suitable competition assay may be used. Useful assays include, forexample, ELISA assays, FACS assays, radioimmunoassays and the like thatquantify the ability of the antibody or homolog to compete with CXCL13or other ligand for binding to human CXCR5. Preferably, the ability ofligand to block binding of labeled human CXCR5 to immobilized antibodyor homolog is measured.

The ability of an antibody or homolog to bind to human CXCR5 can beevaluated by testing the ability thereof to bind to human CXCR5⁺ cells.Suitable CXCR5⁺ cells for use in determining whether a particularantibody or homolog binds to human CXCR5 are mammal tissue culture cellstransformed with DNA encoding full-length human CXCR5 and expressing theCXCR5 on the cell surface or B cell lines.

Binding of the antibody or homolog to the CXCR5⁺ cell can be detected bystaining the cells with a fluorescently-labeled second antibody specificfor immunoglobulins of the same species from which the antibody homologbeing tested is derived. A fluorescence activated cell sorter (“FACS”)can be used to detect and to quantify any binding, see generally,Shapiro, Practical Flow Cytometry, Alan R. Liss, Inc., New York, N.Y.(1985).

Also, the ability of an antibody homolog to block binding of a ligand,such as CXCL13, to human CXCR5 can be determined by preincubating excessligand with CXCR5⁺ cells and quantifying the degree to which the boundligand blocks binding of the antibody or homolog to the cells. Bindingof the antibody homolog to the CXCR5⁺ cells can be quantified by FACSanalysis, using a fluorescently labeled second antibody specific forimmunoglobulins of the same species from which the antibody homologbeing tested is derived. Alternatively, a competition assay can beconfigured using labeled ligand or antibody as known in the art.

Ligand, such as CXCL13, used in the above assays may be provided bycells transformed with the gene for the ligand, or by isolated CXCL13,obtained practicing methods taught herein, or purchased commercially.

To determine whether a particular antibody or homolog causes nosignificant decrease in the number of circulating CXCR5⁺ cells in vivo,the number of circulating CXCR5⁺ cells isolated from a mammal within 24hours after administration of the antibody or homolog to a mammal havingnormal immune function is quantified, and compared to thepre-administration number or the number in a control mammal to whom anisotype-matched antibody or homolog of irrelevant specificity has beenadministered instead of an antibody or homolog of the instant invention.Quantification of CXCR5⁺ cells in animals dosed with a CXCR5 antibody orfunctional portion or derivative thereof may be accomplished, forexample, by staining obtained cells with fluorescently-labeledantibodies that bind the anti-CXCR5 antibodies, as well as labeledantibodies specific for T cells and B cells, followed by FACS analysis.

Antibodies of the instant invention may be described or specified interms of the epitope(s) or portion(s) of CXCR5 to which the antibodyrecognizes or specifically binds. The epitope(s) or polypeptideportion(s) may be specified as described herein, e.g., by N-terminal andC-terminal positions, by size in contiguous amino acid residues,conformational epitopes and so on.

Antibodies of the instant invention may also be described or specifiedin terms of cross-reactivity. Antibodies that bind CXCR5 polypeptides,which have at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 65%, at least 60%, at least 55%, andat least 50% identity (as calculated using methods known in the art anddescribed herein) to CXCR5 are also included in the instant invention.

Antibodies of the instant invention also may be described or specifiedin terms of binding affinity to a CXCR5 of interest. Anti-CXCR5antibodies may bind with a K_(D) of less than about 10⁻⁷ M, less thanabout 10⁻⁶ M, or less than about 10⁻⁵ M. Higher binding affinities in anantibody of interest can be beneficial, such as those with anequilibrium dissociation constant or K_(D) of from about 10⁻⁸ to about10⁻¹⁵ M, from about 10⁻⁸ to about 10⁻¹² M, from about 10⁻⁹ to about10⁻¹¹ M, or from about 10⁻⁸ to about 10⁻¹⁰ M. The invention alsoprovides antibodies that competitively inhibit binding of an antibody toan epitope of the invention as determined by any method known in the artfor determining competitive binding, for example, the immunoassaysdescribed herein. In preferred embodiments, the antibody competitivelyinhibits binding to the epitope by at least 95%, at least 90%, at least85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least50%.

The instant invention also includes conjugates comprising an antibody ofinterest. The conjugates comprise two primary components, an antibody ofinterest and a second component, which may be a cell-binding agent, acytotoxic agent and so on.

As used herein, the term “cell-binding agent” refers to an agent thatspecifically recognizes and binds to a molecule on the cell surface.Thus, the cell-binding agent can be a CD antigen, a pathogen antigen,such as a virus antigen, a differentiation antigen, a cancer antigen, acell-specific antigen, a tissue-specific antigen, an Ig or Ig-likemolecule and so on.

In one embodiment, the cell-binding agent specifically recognizes CXCL13or the complex of CXCR5 and a ligand thereof, such as CXCL13. Theconjugate may be in contact with the target cell for a sufficient periodof time to allow an effector function of the conjugate to act on thecell, and/or to allow the conjugate sufficient time in which to beinternalized by the cell.

Cell-binding agents may be of any type as presently known, or thatbecome known, and includes peptides, non-peptides, saccharides, nucleicacids, ligands, receptors and so on, or combinations thereof. Thecell-binding agent may be any compound that can bind a cell, either in aspecific or non-specific manner. Generally, the agent can be an antibody(especially monoclonal antibodies), lymphokines, hormones, growthfactors, vitamins, nutrient-transport molecules (such as transferrin),or any other cell-binding molecule or substance.

Other examples of cell-binding agents that can be used include:polyclonal antibodies; monoclonal antibodies; and fragments ofantibodies such as F_(ab), F_(ab′), F_((ab′)2) and F_(v) (Parham, J.Immunol. 131:2895-2902 (1983); Spring et al., J. Immunol. 113:470-478(1974); and Nisonoff et al., Arch. Biochem. Biophys. 89: 230-244(1960)).

The second component also can be a cytotoxic agent. The term “cytotoxicagent” as used herein refers to a substance that reduces or blocks thefunction, or growth, of cells and/or causes destruction of cells. Thus,the cytotoxic agent can be a taxol, a maytansinoid, such as DM1 or DM4,CC-1065 or a CC-1065 analog, a ricin, mitomycin C and so on. In someembodiments, the cytotoxic agent, as with any binding agent of aconjugate of the instant invention is covalently attached, directly orvia a cleavable or non-cleavable linker, to an antibody of interest.

Examples of suitable maytansinoids include maytansinol and maytansinolanalogs. Maytansinoids inhibit microtubule formation and are highlytoxic to mammalian cells.

Examples of suitable maytansinol analogues include those having amodified aromatic ring and those having modifications at otherpositions. Such suitable maytansinoids are disclosed in U.S. Pat. Nos.4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929;4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348;4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

Examples of suitable analogues of maytansinol having a modified aromaticring include: (1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared, forexample, by LAH reduction of ansamytocin P2); (2) C-20-hydroxy (orC-20-demethyl)+/− C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016)(prepared, for example, by demethylation using Streptomyces orActinomyces or dechlorination using lithium aluminum hydride (LAH)); and(3) C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No.4,294,757) (prepared by acylation using acyl chlorides).

Examples of suitable analogues of maytansinol having modifications ofother positions include: (1) C-9-SH (U.S. Pat. No. 4,424,219) (preparedby the reaction of maytansinol with H₂S or P₂S₅); (2) C-14-alkoxymethyl(demethoxy/CH₂OR) (U.S. Pat. No. 4,331,598); (3) C-14-hydroxymethyl oracyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No. 4,450,254) (prepared fromNocardia); (4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (preparedby the conversion of maytansinol by Streptomyces); (5) C-15-methoxy(U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewianudiflora); (6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348)(prepared by the demethylation of maytansinol by Streptomyces); and (7)4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titaniumtrichloride/LAH reduction of maytansinol).

The cytotoxic conjugates may be prepared by in vitro methods. To link acytotoxic agent, drug or prodrug to the antibody, commonly, a linkinggroup is used. Suitable linking groups are known in the art and includedisulfide groups, thioether groups, acid labile groups, photolabilegroups, peptidase labile groups and esterase labile groups. For example,conjugates can be constructed using a disulfide exchange reaction or byforming a thioether bond between an antibody of interest and the drug orprodrug.

As discussed above, the instant invention provides isolated nucleic acidsequences encoding an antibody or functional variant thereof asdisclosed herein, vector constructs comprising a nucleotide sequenceencoding the CXCR5-binding polypeptides of the present invention, hostcells comprising such a vector, and recombinant techniques for theproduction of the polypeptide.

The vector normally contains components known in the art and generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker or selectiongenes, sequences facilitating and/or enhancing translation, an enhancerelement and so on. Thus, the expression vectors include a nucleotidesequence operably linked to such suitable transcriptional ortranslational regulatory nucleotide sequences such as those derived frommammalian, microbial, viral or insect genes. Examples of additionalregulatory sequences include operators, mRNA ribosomal binding sites,and/or other appropriate sequences which control transcription andtranslation, such as initiation and termination thereof. Nucleotidesequences are “operably linked” when the regulatory sequencefunctionally relates to the nucleotide sequence for the appropriatepolypeptide. Thus, a promoter nucleotide sequence is operably linked to,e.g., the antibody heavy chain sequence if the promoter nucleotidesequence controls the transcription of that nucleotide sequence.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with antibody heavy and/or light chain sequencescan be incorporated into expression vectors. For example, a nucleotidesequence for a signal peptide (secretory leader) may be fused in-frameto the polypeptide sequence so that the antibody is secreted to theperiplasmic space or into the medium. A signal peptide that isfunctional in the intended host cells enhances extracellular secretionof the appropriate antibody or portion thereof. The signal peptide maybe cleaved from the polypeptide on secretion of antibody from the cell.Examples of such secretory signals are well known and include, e.g.,those described in U.S. Pat. Nos. 5,698,435; 5,698,417; and 6,204,023.

The vector may be a plasmid, a single-stranded or double-stranded viralvector, a single-stranded or double-stranded RNA or DNA phage vector, aphagemid, a cosmid or any other carrier of a transgene of interest. Suchvectors may be introduced into cells as polynucleotides by well knowntechniques for introducing DNA and RNA into cells. The vectors, in thecase of phage and viral vectors also may be introduced into cells aspackaged or encapsulated virus by well known techniques for infectionand transduction. Viral vectors may be replication competent orreplication defective. In the latter case, viral propagation generallywill occur only in complementing host cells and using plural vectorscarrying the various virus components necessary to produce a particle.Cell-free translation systems may also be employed to produce theprotein using RNAs derived from the present DNA constructs (see, e.g.,WO 86/05807 and WO 89/01036; and U.S. Pat. No. 5,122,464).

The antibodies of the present invention can be expressed from anysuitable host cell. Examples of host cells useful in the instantinvention include prokaryotic, yeast or higher eukaryotic cells andinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, Serratia, and Shigella, as well as Bacilli, Pseudomonas andStreptomyces) transformed with recombinant bacteriophage DNA, plasmidDNA or cosmid DNA expression vectors containing the antibody codingsequences of interest; yeast (e.g., Saccharomyces, Pichia,Actinomycetes, Kluyveromyces, Schizosaccharomyces, Candida, Trichoderma,Neurospora, and filamentous fungi, such as Neurospora, Penicillium,Tolypocladium and Aspergillus) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,Baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; or tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293 or 3T3 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; or the vaccinia virus 7.5K promoter).

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids, such as pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec,Madison, Wis.), pET (Novagen, Madison, Wis.) and the pRSET (Tnvitrogen,Carlsbad, Calif.) series of vectors (Studier, J Mol Biol 219:37 (1991);and Schoepfer, Gene 124:83 (1993)). Promoter sequences commonly used forrecombinant prokaryotic host cell expression vectors include T7,(Rosenberg et al., Gene 56:125 (1987)), β-lactamase (penicillinase),lactose promoter system (Chang et al., Nature 275:615 (1978); andGoeddel et al., Nature 281:544 (1979)), tryptophan (trp) promoter system(Goeddel et al., Nucl Acids Res 8:4057 (1980)), and tac promoter(Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory (1990)).

Yeast vectors will often contain an origin of replication sequence, suchas from a 2μ yeast plasmid, an autonomously replicating sequence (ARS),a promoter region, sequences for polyadenylation, sequences fortranscription termination and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J BiolChem 255:2073 (1980)) or other glycolytic enzymes (Holland et al.,Biochem 17:4900 (1978)) such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Fleer et al., Gene 107:285 (1991).Other suitable promoters and vectors for yeast and yeast transformationprotocols are well known in the art. Yeast transformation protocols arewell known. One such protocol is described by Hinnen et al., Proc NatlAcad Sci 75:1929 (1978), which selects for Trp⁺ transformants in aselective medium.

Any eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells (Luckow et al., Bio/Technology 6:47 (1988); Miller et al.,Genetic Engineering, Setlow et al., eds., vol. 8, pp. 277-9, PlenumPublishing (1986); and Maeda et al., Nature 315:592 (1985)). Forexample, Baculovirus systems may be used for production of heterologousproteins. In an insect system, Autographa californica nuclearpolyhedrosis virus (AcNPV) may be used as a vector to express foreigngenes. The virus grows in Spodoptera frugiperda cells. The antibodycoding sequence may be cloned under control of an AcNPV promoter (forexample the polyhedrin promoter). Other hosts that have been identifiedinclude Aedes, Drosophila melanogaster and Bombyx mori. A variety ofviral strains for transfection are publicly available, e.g., the L-1variant of AcNPV and the Bm-5 strain of Bombyx mori NPV. Moreover, plantcell cultures of cotton, corn, potato, soybean, petunia, tomato, andtobacco and also be utilized as hosts as known in the art.

Vertebrate cells, and propagation of vertebrate cells, in culture(tissue culture) can be a routine procedure, although fastidious celllines do exist which require, for example, a specialized medium withunique factors, feeder cells and so on, see Tissue Culture, Kruse etal., eds., Academic Press (1973). Examples of useful mammal host celllines are monkey kidney; human embryonic kidney line; baby hamsterkidney cells; Chinese hamster ovary cells/−DHFR (CHO, Urlaub et al.,Proc Natl Acad Sci USA 77:4216 (1980)); mouse sertoli cells; humancervical carcinoma cells (for example, HeLa); canine kidney cells; humanlung cells; human liver cells; mouse mammary tumor; and NS0 cells.

Host cells are transformed with vectors for antibody production andcultured in conventional nutrient medium containing growth factors,vitamins, minerals and so on, as well as inducers appropriate for thecells and vectors used. Commonly used promoter sequences and enhancersequences are derived from polyoma virus, Adenovirus 2, Simian virus 40(SV40) and human cytomegalovirus (CMV). DNA sequences derived from theSV40 viral genome may be used to provide other genetic elements forexpression of a structural gene sequence in a mammalian host cell, e.g.,SV40 origin, early and late promoter, enhancer, splice andpolyadenylation sites. Viral early and late promoters are particularlyuseful because both are easily obtained from a viral genome as afragment which may also contain a viral origin of replication. Exemplaryexpression vectors for use in mammalian host cells are commerciallyavailable.

Commercially available medium such as Ham's F10, Minimal EssentialMedium (MEM), RPMI-1640 and Dulbecco's Modified Eagle's Medium (DMEM)are suitable for culturing host cells. In addition, any of the mediadescribed in Ham et al., Meth Enzymol 58:44 (1979) and Barnes et al.,Anal Biochem 102:255 (1980), and in U.S. Pat. Nos. 4,767,704; 4,657,866;4,560,655; 5,122,469; 5,712,163; or 6,048,728 may be used as a culturemedium for the host cells. Any of those media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin or epidermal growth factor), salts (such as chlorides, suchas sodium, calcium or magnesium chloride; and phosphates), buffers (suchas HEPES), nucleotides (such as adenosine and thymidinc), antibiotics,trace elements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range) and glucose or an equivalentenergy source. Any other necessary supplements may be included atappropriate concentrations, as a design choice. The culture conditions,such as temperature, pH and the like, are as known in the artappropriate for the cell and to enable the desired expression of thetransgene.

The polynucleotides of interest may be obtained, and the nucleotidesequence of the polynucleotides determined, by any method known in theart. For example, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,Bio/Techniques 17:242 (1994)) and then amplifying the ligatedoligonucleotides, for example, by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid of a cell expressing same. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be obtained from a suitable source, such as alibrary, which may be one specific for antibody-producing cells, such ashybridoma cells selected to express an antibody of the invention.Suitable primers can be configured for PCR amplification. Amplifiednucleic acids generated by PCR may then be cloned into replicablecloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody are determined, the nucleotide sequence of the antibody maybe manipulated to obtain the equivalents of interest described hereinusing methods known in the art for manipulating nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR etc.(see, for example, Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory (1990); and Ausubel etal., eds., Current Protocols in Molecular Biology, John Wiley & Sons(1998) to generate antibodies having a different amino acid sequence,for example, to create amino acid substitutions, deletions and/orinsertions.

The amino acid sequence of the heavy and/or light chain variable domainmay be inspected to identify the sequences of the CDRs by well knownmethods, e.g., by comparison to known amino acid sequences of otherheavy and light chain variable regions to determine the regions ofsequence hypervariability. Using routine recombinant DNA techniques, oneor more of the CDRs may be inserted within framework regions, e.g., intohuman framework regions to humanize a non-human antibody, as describedsupra. The polynucleotide of interest generated by the combination ofthe framework regions and one or more CDRs encodes an antibody thatspecifically binds CXCR5, or at least the ED domain thereof. Forexample, such methods may be used to make amino acid substitutions ordeletions of one or more variable region cysteine residues participatingin an intrachain disulfide bond to generate antibody molecules lackingone or more intrachain disulfide bonds.

The antibodies or antibody fragments of the invention can be used todetect CXCR5, and hence cells expressing CXCR5, in a biological samplein vitro or in vivo. In one embodiment, the anti-CXCR5 antibody of theinvention is used to determine the presence and the level of CXCR5 in atissue or in cells derived from the tissue. The levels of CXCR5 in thetissue or biopsy can be determined, for example, in an immunoassay withthe antibodies or antibody fragments of the invention. The tissue orbiopsy thereof can be frozen or fixed. The same or other methods can beused to determine other properties of CXCR5, such as the level thereof,cellular localization, mRNA levels, mutations thereof and so on.

The above-described method can be used, for example, to diagnose acancer in a subject known to be or suspected to have a cancer, whereinthe level of CXCR5 measured in said patient is compared with that of anormal reference subject or standard. The assay of interest also can beused to diagnose arthritis or other autoimmune diseases characterized byB cell infiltration and concentration, along with development ofdifferentiated lymphoid tissue.

The instant invention further provides for monoclonal antibodies,humanized antibodies and epitope-binding fragments thereof that arefurther labeled for use in research or diagnostic applications. In someembodiments, the label is a radiolabel, a fluorophore, a chromophore, animaging agent or a metal ion.

A method for diagnosis is also provided in which said labeled antibodiesor epitope-binding fragments thereof are administered to a subjectsuspected of having a cancer, arthritis, autoimmune diseases or otherCXCR5 disease, and the distribution of the label within the body of thesubject is measured or monitored.

The antibody and fragments thereof of the instant invention may be usedas affinity purification agents. In that process, the antibodies areimmobilized on a solid phase, such as a dextran or agarose resin orfilter paper, using methods known in the art. The immobilized antibodyis contacted with a sample containing CXCR5 or cells carrying same to bepurified, and thereafter the support is washed with a suitable solventthat will remove substantially all the material in the sample except theCXCR5 or cell to be purified, which is bound to the immobilized antibodyof interest. Finally, the support is washed with another suitablesolvent, such as glycine buffer, pH 5.0, that will release the CXCR5 orcell from the antibody of interest.

For diagnostic applications, the antibody of interest typically will belabeled with a detectable moiety. Numerous labels are available whichcan be generally grouped into the following categories: (a)radioisotopes, such as ³⁶S, ¹⁴C, ¹²⁵I, ³H and ¹³¹I (The antibody can belabeled with the radioisotope using a techniques described in CurrentProtocols in Immunology, vol. 12, Coligen et al., ed.,Wiley-Interscience, New York (1991), for example, and radioactivity canbe measured using scintillation counting); (b) fluorescent labels, suchas rare earth chelates (europium chelates), fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, lissamine,phycoerythrin and Texas Red, the fluorescent labels can be conjugated tothe antibody using a technique disclosed in Current Protocols inImmunology, supra, for example, where fluorescence can be quantifiedusing a fluorimeter; and (c) various enzyme substrate labels areavailable (U.S. Pat. No. 4,275,149 provides a review), the enzymegenerally catalyzes a chemical alteration of the chromogenic substratewhich can be measured using various techniques, for example, the enzymemay catalyze a color change in a substrate, which can be measuredspectrophotometrically, or the enzyme may alter the fluorescence orchemiluminescence of the substrate. Techniques for quantifying a changein fluorescence are known, for example, using a luminometer, or thelabel donates energy to a fluorescent acceptor. Examples of enzymaticlabels include luciferases (e.g., firefly luciferase and bacterialluciferase; U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase,such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharidc oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Meth Enz, ed. Langone & Van Vunakis, Academic Press, New York, 73(1981).

When such labels are used, suitable substrates are available, such as:(i) for horseradish peroxidase with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor (e.g.,orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB)); (ii) for alkaline phosphatase (AP) withp-nitrophenyl phosphate as the chromogenic substrate; and (iii)β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or a fluorogenic substrate such as4-methylumbelliferyl-β-D-galactosidase.

Other enzyme-substrate combinations are available to those skilled inthe art. For a general review, see U.S. Pat. Nos. 4,275,149 and4,318,980.

Sometimes, the label is indirectly conjugated with the antibody. Forexample, the antibody can be conjugated with biotin and any of thereporters mentioned above can be conjugated with avidin, or vice versa.Biotin binds selectively to avidin and thus, the label can be conjugatedwith the antibody in that indirect manner. Alternatively, to achieveindirect conjugation of the label, the antibody is conjugated with asmall hapten (e.g., digoxin) and one of the different types of labels orreporters mentioned above is conjugated with an anti-digoxin antibody.Thus, indirect conjugation of the label with the antibody or mutein canbe achieved using a second antibody.

In another embodiment of the invention, the antibody need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody, another form of a second antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample for binding with a limited amount ofantibody. The amount of antigen in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test sample thatare bound to the antibodies may conveniently be separated from thestandard and test sample which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, determinant or epitope, ofthe target to be detected. In a sandwich assay, the test sample to beanalyzed is bound by a first antibody which is immobilized directly orindirectly on a solid support, and thereafter a second antibody directlyor indirectly labeled binds to the bound test sample, thus forming aninsoluble three-part complex, see e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody or other suitable member of the binding pair (antibody/antigen,receptor/ligand, enzyme/substrate, for example) that is labeled with adetectable moiety (indirect sandwich assay). For example, one type ofsandwich assay is an ELISA assay, in which case the detectable moiety isan enzyme.

For immunohistochemistry, the cell or tissue sample may be fresh orfrozen or may be embedded in paraffin and fixed with a preservative suchas formalin, for example.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody mutant is labeled with a radionucleotide (suchas ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the sites expressingCXCR5 can be localized using immunoscintography.

The instant invention also includes kits, e.g., comprising an antibody,fragment thereof, homolog, derivative thereof and so on, such as alabeled or cytotoxic conjugate, and instructions for the use of theantibody, conjugate for killing particular cell types and so on. Theinstructions may include directions for using the antibody, conjugateand so on in vitro, in vivo or ex vivo. The antibody can be in liquidform or as a solid, generally lyophilized. The kit can contain suitableother reagents, such as a buffer, a reconstituting solution and othernecessary ingredients for the intended use. A packaged combination ofreagents in predetermined amounts with instructions for use thereof,such as for a therapeutic use of for performing a diagnostic assay iscontemplated. Where the antibody is labeled, such as with an enzyme, thekit can include substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied toprovide for concentrates of a solution of a reagent, which provides userflexibility, economy of space, economy of reagents and so on. Thereagents may be provided as dry powders, usually lyophilized, includingexcipients, which on dissolution provide a reagent solution having theappropriate concentration.

The antibodies of the present invention may be used to treat a mammal.In one embodiment, the antibody or equivalent of interest isadministered to a nonhuman mammal for the purposes of obtainingpreclinical data, for example. Exemplary nonhuman mammals to be treatedinclude nonhuman primates, dogs, cats, rodents and other mammals inwhich preclinical studies are performed. Such mammals may be establishedanimal models for a disease to be treated with the antibody, or may beused to study toxicity of the antibody of interest. In each of thoseembodiments, dose escalation studies may be performed in the mammal.

An antibody, with or without a second component, such as a therapeuticmoiety conjugated to same, administered alone or in combination withcytotoxic factor(s) can be used as a therapeutic. The present inventionis directed to antibody-based therapies which involve administeringantibodies of the invention to an animal, a mammal, or a human, fortreating a CXCR5-mediated disease, disorder or condition. The animal orsubject may be a mammal in need of a particular treatment, such as amammal having been diagnosed with a particular disorder, e.g., onerelating to CXCR5. Antibodies directed against CXCR5 are useful, forexample, for prophylaxis or treatment of arthritis, inflammatorydiseases, in general, graft rejection, cancer and autoimmune disorders.For example, by administering a therapeutically acceptable dose of ananti-CXCR5 antibody of the instant invention, or a cocktail of aplurality of the instant antibodies or equivalents thereof, or incombination with other antibodies of varying sources, disease symptomsmay be ameliorated or prevented in the treated mammal, particularlyhumans.

Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs, equivalentsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention as described herein (including fragments,analogs and derivatives thereof) and anti-idiotypic antibodies asdescribed herein. The antibodies of the invention can be used to treat,inhibit or prevent diseases, disorders or conditions associated withaberrant expression and/or activity of CXCR5, including, but not limitedto, any one or more of the diseases, disorders, or conditions describedherein. The treatment and/or prevention of diseases, disorders orconditions associated with aberrant expression and/or activity of CXCR5includes, but is not limited to, alleviating at least one symptomassociated with those diseases, disorders, or conditions. Antibodies ofthe invention may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein. The term“physiologically acceptable,” “pharmacologically acceptable” and so onmean approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals and more particularly inhumans.

The anti-CXCR5 antibody can be administered to a mammal in anyacceptable manner. Methods of introduction include, but are not limitedto, parenteral, subcutaneous, intraperitoneal, intrapulmonary,intranasal, epidural, inhalation and oral routes, and if desired forimmunosuppressive treatment, intralesional administration. Parenteralinfusions include intramuscular, intradermal, intravenous, intraarterialor intraperitoneal administration. The antibodies or compositions may beadministered by any convenient route, for example, by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the therapeutic antibodies or compositions of theinvention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir. Inaddition, the antibody is suitably administered by pulse infusion,particularly with declining doses of the antibody. Preferably the dosingis given by injection, preferably intravenous or subcutaneousinjections, depending, in part, on whether the administration is briefor chronic.

Various other delivery systems are known and can be used to administeran antibody of the present invention, including, e.g., encapsulation inliposomes, microparticles, microcapsules (see Langer, Science 249:1527(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer; Lopez-Berestein et al., eds., p. 353-365 (1989); andLopez-Berestein, ibid., p. 317-327) and recombinant cells capable ofexpressing the compound; receptor-mediated endocytosis (see, e.g., Wu etal., J Biol Chem 262:4429 (1987)); construction of a nucleic acid aspart of a retroviral or other vector etc.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coascervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nanoparticles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, A. Osal, Ed. (1980).

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. Theantibody may also be administered into the lungs of a patient in theform of a dry powder composition, see e.g., U.S. Pat. No. 6,514,496.

In a specific embodiment, it may be desirable to administer thetherapeutic antibodies or compositions of the invention locally to thearea in need of treatment; that may be achieved by, for example, and notby way of limitation, local infusion, topical application, by injection,by means of a catheter, by means of a suppository or by means of animplant, said implant being of a porous, non-porous or gelatinousmaterial, including membranes, such as sialastic membranes or fibers.Preferably, when administering an antibody of the invention, care istaken to use materials to which the protein does not absorb or adsorb.

In yet another embodiment, the antibody can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,Science 249:1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); and Saudek et al., N Engl J Med321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer et al., eds.,CRC Press (1974); Controlled Drug Bioavailability, Drug Product Designand Performance, Smolen et al., eds., Wiley (1984); Ranger et al., JMacromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al.,Science 228:190 (1985); During et al., Ann Neurol 25:351 (1989); andHoward et al., J Neurosurg 71:105 (1989)). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget.

Therapeutic formulations of the polypeptide or antibody may be preparedfor storage as lyophilized formulations or aqueous solutions by mixingthe polypeptide having the desired degree of purity with optional“pharmaceutically acceptable” carriers, diluents, excipients orstabilizers typically employed in the art, i.e., buffering agents,stabilizing agents, preservatives, isotonifiers, non-ionic detergents,antioxidants and other miscellaneous additives, see Remington'sPharmaceutical Sciences, 16th ed., Osol, ed. (1980). Such additives aregenerally nontoxic to the recipients at the dosages and concentrationsemployed, hence, the excipients, diluents, carriers and so on arepharmaceutically acceptable.

An “isolated” or “purified” antibody is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourceor medium from which the protein is derived, or substantially free ofchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof an antibody in which the polypeptide/protein is separated fromcellular components of the cells from which same is isolated orrecombinantly produced. Thus, an antibody that is substantially free ofcellular material includes preparations of the antibody having less thanabout 30%, 20%, 10%, 5%, 2.5% or 1%, (by dry weight) of contaminatingprotein. When the antibody is recombinantly produced, it is alsopreferably substantially free of culture medium, i.e., culture mediumrepresents less than about 20%, 10%, 5%, 2.5% or 1% of the volume of theprotein preparation. When antibody is produced by chemical synthesis, itis preferably substantially free of chemical precursors or otherchemicals and reagents, i.e., the antibody of interest is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly, such preparations of the antibodyhave less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemicalprecursors or compounds other than antibody of interest. In a preferredembodiment of the present invention, antibodies are isolated orpurified.

As used herein, the phrase “low to undetectable levels of aggregation”refers to samples containing no more than 5%, no more than 4%, no morethan 3%, no more than 2%, no more than 1% and often no more than 0.5%aggregation, by weight protein, as measured by, for example, highperformance size exclusion chromatography (HPSEC).

As used herein, the term “low to undetectable levels of fragmentation”refers to samples containing equal to or more than 80%, 85%, 90%, 95%,98% or 99%, of the total protein, for example, in a single peak, asdetermined by HPSEC, or in two (2) peaks (heavy chain and light chain)by, for example, reduced capillary gel electrophoresis (rCGE) andcontaining no other single peaks having more than 5%, more than 4%, morethan 3%, more than 2%, more than 1% or more than 0.5% of the totalprotein, each. The rCGE as used herein refers to capillary gelelectrophoresis under reducing conditions sufficient to reduce disulfidebonds in an antibody or antibody-type or derived molecule.

As used herein, the terms “stability” and “stable” in the context of aliquid formulation comprising a CXCR5 antibody or binding fragmentthereof refer to the resistance of the antibody or antigen-bindingfragment thereof in the formulation to thermal and chemical unfolding,aggregation, degradation or fragmentation under given manufacture,preparation, transportation and storage conditions. The “stable”formulations of the invention retain biological activity equal to ormore than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% under given manufacture,preparation, transportation and storage conditions. The stability ofsaid antibody preparation can be assessed by degrees of aggregation,degradation or fragmentation by methods known to those skilled in theart, including, but not limited to, rCGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and HPSEC, compared to areference.

The term, “carrier,” refers to a diluent, adjuvant, excipient or vehiclewith which the therapeutic is administered. Such physiological carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a suitablecarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions also can be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. The compositionscan take the form of solutions, suspensions, emulsion, tablets, pills,capsules, powders, sustained-release formulations, depots and the like.The composition can be formulated as a suppository, with traditionalbinders and carriers such as triglycerides. Oral formulations caninclude standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate etc. Examples of suitable carriers are described in“Remington's Pharmaceutical Sciences,” Martin. Such compositions willcontain an effective amount of the antibody, preferably in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the patient. As known in the art, theformulation will be constructed to suit the mode of administration.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. Buffers are preferably present at aconcentration ranging from about 2 mM to about 50 mM. Suitable bufferingagents for use with the instant invention include both organic andinorganic acids, and salts thereof, such as citrate buffers (e.g.,monosodium citrate-disodium citrate mixture, citric acid-trisodiumcitrate mixture, citric acid-monosodium citrate mixture etc.), succinatebuffers (e.g., succinic acid-monosodium succinate mixture, succinicacid-sodium hydroxide mixture, succinic acid-disodium succinate mixtureetc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture,tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxidemixture etc.), fumarate buffers (e.g., fumaric acid-monosodium fumaratemixture, fumaric acid-disodium fumarate mixture, monosodiumfumarate-disodium fumarate mixture etc.), gluconate buffers (e.g.,gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxidemixture, gluconic acid-potassium gluconate mixture etc.), oxalatebuffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodiumhydroxide mixture, oxalic acid-potassium oxalate mixture etc.), lactatebuffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodiumhydroxide mixture, lactic acid-potassium lactate mixture etc.) andacetate buffers (e.g., acetic acid-sodium acetate mixture, aceticacid-sodium hydroxide mixture etc.). Phosphate buffers, carbonatebuffers, histidine buffers, trimethylamine salts such as Tris, HEPES andother such known buffers can be used.

Preservatives may be added to retard microbial growth, and may be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, m-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzyaconium halides (e.g., chloride, bromide and iodide),hexamethonium chloride, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are present to ensure physiological isotonicity of liquidcompositions of the instant invention and include polhydric sugaralcohols, preferably trihydric or higher sugar alcohols, such asglycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.Polyhydric alcohols can be present in an amount of between about 0.1% toabout 25%, by weight, preferably 1% to 5% taking into account therelative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols;amino acids, such as arginine, lysine, glycine, glutamine, asparagine,histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamicacid, threonine etc., organic sugars or sugar alcohols, such as lactose,trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol,ribitol, myoinisitol, galactitol, glycerol and the like, includingcyclitols such as inositol; polyethylene glycol; amino acid polymers;sulfur containing reducing agents, such as urea, glutathione, thiocticacid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodiumthiosulfate; low molecular weight polypeptides (i.e., <10 residues);proteins, such as human serum albumin, bovine serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone,saccharides, monosaccharides, such as xylose, mannose, fructose,glucose; disaccharides, such as lactose, maltose and sucrose;trisaccharides such as raffinose; polysaccharides such as dextran and soon. Stabilizers are present in the range from 0.1 to 10,000 w/w per partof active protein.

Additional miscellaneous excipients include bulking agents, (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine or vitamin E) and cosolvents.

The formulation herein also may contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely impact each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules suitably are present in combination in amountsthat are effective for the purpose intended.

As used herein, the term “surfactant” refers to organic substanceshaving amphipathic structures, namely, are composed of groups ofopposing solubility tendencies, typically an oil-soluble hydrocarbonchain and a water-soluble ionic group. Surfactants can be classified,depending on the charge of the surface-active moiety, into anionic,cationic and nonionic surfactants. Surfactants often are used aswetting, emulsifying, solubilizing and dispersing agents for variouspharmaceutical compositions and preparations of biological materials.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic agent, as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stresseswithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80 etc.), polyoxamers (184, 188etc.), Pluronic® polyols and polyoxyethylene sorbitan monoethers(TWEEN-20°, TWEEN-80° etc.). Non-ionic surfactants may be present in arange of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

As used herein, the term, “inorganic salt,” refers to any compound,containing no carbon, that result from replacement of part or all of theacid hydrogen or an acid by a metal or a group acting like a metal, andoften are used as a tonicity adjusting compound in pharmaceuticalcompositions and preparations of biological materials. The most commoninorganic salts are NaCl, KCl, NaH₂PO₄ etc.

The present invention provides liquid formulations of ananti-CXCR5-binding compound or fragment thereof, having a pH rangingfrom about 5.0 to about 7.0, or about 5.5 to 6.5, or about 5.8 to about6.2, or about 6.0.

The instant invention encompasses liquid formulations having stabilityat temperatures found in a commercial refrigerator and freezer found inthe office of a physician or laboratory, such as from about −20° C. toabout 5° C., said stability assessed, for example, by high performancesize exclusion chromatography (HPSEC), for storage purposes, such as forabout 60 days, for about 120 days, for about 180 days, for about a year,for about 2 years or more. The liquid formulations of the presentinvention also exhibit stability, as assessed, for example, by HSPEC, atroom temperatures, for a at least a few hours, such as one hour, twohours or about three hours prior to use.

The term “small molecule” and analogous terms include, but are notlimited to, peptides, peptidomimetics, amino acids, amino acidanalogues, polynucleotides, polynucleotide analogues, nucleotides,nucleotide analogues, organic or inorganic compounds (i.e., includingheterorganic and/or ganometallic compounds) having a molecular weightless than about 10,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 5,000 grams per mole, organicor inorganic compounds having a molecular weight less than about 1,000grams per mole, organic or inorganic compounds having a molecular weightless than about 500 grams per mole, and salts, esters, and otherpharmaceutically acceptable forms of such compounds.

Thus, in the case of cancer, for example, the antibodies of theinvention may be administered alone or in combination with other typesof cancer treatments, including conventional chemotherapeutic agents(paclitaxel, carboplatin, cisplatin and doxorubicin), anti-EGFR agents(gefitinib, erlotinib and cetuximab), anti-angiogenesis agents(bevacizumab and sunitinib), as well as immunomodulating agents, such asinterferon α and thalidomide.

In another embodiment, in the case of rheumatic diseases, such asrheumatoid arthritis (RA), a combination therapy can be used comprisinga CXCR-binding molecule of interest. For example, a humanized CXCR5antibody can be dosed with a small molecule, such as a disease modifyingantirheumatic drug, including, but not limited to, for example,methotrexate and pyridine synthesis inhibitors, such as, leflunomide(Mader & Keystone, J Rheum 34 Supp (16-24) 2007, Gaffo et al., Am JHealth Syst Pharm 63:2451-2465, 2006).

Because various forms of a CXCR5-binding molecule of interest can benon-B cell-depleting, the instant molecule can be combined with otherdrugs having overlapping mechanisms of action to yield an additive orsynergistic endpoint. Hence, for example, a second drug can be one whichacts at the level of a cytokine, in the T cell axis and so on.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the treatment, management oramelioration of a disease, disorder, malady and the like associated withaberrant CXCR5 and/or CXCL13 metabolism and activity. That can bemanifest in abnormal B cell levels or B cell activity. Also included areknown compounds with a pharmacologic effect in treating a disorder andso on that is associated with aberrant CXCR5 and/or CXCL13 metabolismand activity.

In addition, the antibodies of the instant invention may be conjugatedto various effector molecules such as heterologous polypeptides, drugs,radionucleotides or toxins, see, e.g., WO 92/08495; WO 91/14438; WO89/12624; U.S. Pat. No. 5,314,995; and EPO 396,387. An antibody orfragment thereof may be conjugated to a therapeutic moiety such as acytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive metal ion (e.g., a emitters such as, for example,²¹³Bi). A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include paclitaxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicine, doxorubicin,daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol and puromycin and analogs orhomologues thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil and decarbazine), alkylating agents (e.g.,mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin, daunomycin anddoxorubicin), antibiotics (e.g., dactinomycin, actinomycin, bleomycin,mithramycin and anthramycin (AMC)), and anti-mitotic agents (e.g.,vincristine and vinblastine).

Techniques for conjugating such a therapeutic moiety to antibodies arewell known, see, e.g., Anion et al., in Monoclonal Antibodies and CancerTherapy, Reisfeld et al. (eds.), p. 243-56 Alan R. Liss (1985);Hellstrom et al., in Controlled Drug Delivery, 2nd ed., Robinson et al.,eds., p. 623-53, Marcel Dekker (1987); Thorpe, in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al., eds., p.475-506 (1985); Monoclonal Antibodies For Cancer Detection and Therapy,Baldwin et al., eds., p. 303-16, Academic Press (1985); and Thorpe, etal., Immunol Rev 62:119 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate,such as a bifunctional antibody, see, e.g., U.S. Pat. No. 4,676,980.

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-α, TNF-β, AIM I (WO 97/33899),AIM II (WO 97/34911), Fas ligand (Takahashi et al., Int Immunol, 6:1567(1994)), VEGF (WO 99/23105); a thrombotic agent; an anti-angiogenicagent, e.g., angiostatin or endostatin; or biological response modifierssuch as, for example, lymphokines, interleukin-1 (IL-1), interleukin-2(IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (GCSF) or othergrowth factors.

The formulations to be used for in vivo administration must be sterile.That can be accomplished, for example, by filtration through sterilefiltration membranes. For example, the liquid formulations of thepresent invention may be sterilized by filtration using a 0.2 μm or a0.22 μm filter.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films or matrices. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethylmethacrylate), poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers (such as injectable microspherescomposed of lactic acid-glycolic acid copolymer) andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. Rational strategies can be devised for stabilization dependingon the mechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S-S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulthydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, amino acid substitutionand developing specific polymer matrix compositions.

The antibody, or variant thereof, composition will be formulated, dosedand administered in a manner consistent with good medical practice.Factors for consideration in this context include the particulardisorder being treated, the particular mammal being treated, theclinical condition of the individual patient, the cause of the disorder,the site of delivery of the agent, the method of administration, thescheduling of administration, and other factors known to medicalpractitioners. The “therapeutically effective amount” of the antibody orvariant to be administered will be governed by such considerations, andcan be the minimum amount necessary to prevent, ameliorate or treat aCXCR5 disease, condition or disorder.

The antibody, or variant thereof, optionally is formulated with one ormore agents currently used to prevent or treat the disorder in question.The effective amount of such other agents depends on the amount ofantibody present in the formulation, the type of disorder or treatmentand other factors discussed above. These are generally used in the samedosages and with administration routes as used hereinbefore or aboutfrom 1 to 99% of the heretofore employed dosages.

As used herein, the term “effective amount” refers to the amount of atherapy (e.g., a prophylactic or therapeutic agent), which is sufficientto reduce the severity and/or duration of a CXCR5 disease, ameliorateone or more symptoms thereof, prevent the advancement of a CXCR5 diseaseor cause regression of a CXCR5 disease, or which is sufficient to resultin the prevention of the development, recurrence, onset, or progressionof a CXCR5 disease or one or more symptoms thereof, or enhance orimprove the prophylactic and/or therapeutic effect(s) of another therapy(e.g., another therapeutic agent) useful for treating a CXCR5 disease.For example, a treatment of interest can reduce elevated B cell levels,based on baseline or a normal level, by at least 5%, preferably at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 100%. In anotherembodiment, an effective amount of a therapeutic or a prophylactic agentreduces the symptoms of a CXCR5 disease, such as arthritis or graftrejection by at least 5%, preferably at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 100%. Also used herein as an equivalent is theterm, “therapeutically effective amount.”

The amount of therapeutic polypeptide, antibody or fragment thereofwhich will be effective in the use or treatment of a particular disorderor condition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. Where possible, adose-response curve and the pharmaceutical compositions of the inventioncan be first derived in vitro. If a suitable animal model system isavailable, again a dose-response curve can be obtained and used toextrapolate a suitable human dose practicing methods known in the art.However, based on common knowledge of the art, a pharmaceuticalcomposition effective in promoting a diminution of an inflammatoryeffect, for example, may provide a local therapeutic agent concentrationof between about 5 and 20 ng/ml, and, preferably, between about 10 and20 ng/ml. In an additional specific embodiment of the invention, apharmaceutical composition effective in ameliorating the growth andsurvival of cells responsible for B cell-dependent autoimmunemanifestations or graft rejection may provide a local therapeutic agentconcentration of between about 10 ng/ml and about 100 ng/ml.

In a preferred embodiment, an aqueous solution of therapeuticpolypeptide, antibody or fragment thereof can be administered bysubcutaneous injection. Each dose may range from about 0.5 mg to about50 mg per kilogram of body weight, or more preferably, from about 3 mgto about 30 mg per kilogram body weight. The dosage can be ascertainedempirically for the particular disease, patient population, mode ofadministration and so on, practicing pharmaceutics methods known in theart.

The dosing schedule for subcutaneous administration may vary from once aweek to daily depending on a number of clinical factors, including thetype of disease, severity of disease and the sensitivity of the subjectto the therapeutic agent.

The instant invention provides methods for preparing liquid formulationsof the antibody or CXCR5-binding fragment thereof, said methodscomprising concentrating a fraction of purified antibody to a finalconcentration of about 15 mg/ml, about 20 mg/ml, about 30 mg/ml, about40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80mg/ml, about 90 mg/ml, about 100 mg/ml, about 200 mg/ml, about 250mg/ml, about 300 mg/ml or more using, for example, a semi-permeablemembrane with an appropriate molecular weight (mw) cutoff (e.g., 30K_(D) cutoff for F_((ab′)2) fragments thereof; and 10 K_(D) cutoff forF_(ab) fragments) and, optionally, diafiltering the concentratedantibody fraction into the formulation buffer using the same membrane.

In addition, the present invention also encompasses stable, such asK_(D) stable, liquid formulations of the products of interest that haveimproved half-life in vivo. Thus, the antibody of interest has ahalf-life in a subject, preferably a human, of greater than 3 days,greater than 7 days, greater than 10 days, greater than 15 days, greaterthan 25 days, greater than 30 days, greater than 35 days, greater than40 days, greater than 45 days, greater than 2 months, greater than 3months, greater than 4 months, greater than 5 months or more.

To prolong the serum circulation of an antibody in vivo, varioustechniques can be used. For example, inert polymer molecules, such ashigh molecular weight polyethylene glycol (PEG), can be attached to anantibody with or without a multifunctional linker either throughsite-specific conjugation of the PEG to the N-terminus or to theC-terminus of the antibody or via ε amino groups present on lysineresidues. Linear or branched polymer derivatization that results inminimal loss of biological activity can be used. The degree ofconjugation can be closely monitored by SDS-PAGE and mass spectrometryto ensure proper conjugation of PEG molecules to the antibodies.Unreacted PEG can be separated from antibody-PEG conjugates bysize-exclusion or by ion exchange chromatography. PEG-derivatizedantibodies can be tested for binding activity as well as for in vivoefficacy using methods known to those of skilled in the art, forexample, by immunoassays described herein.

An antibody having an increased half-life in vivo can also be generatedby introducing one or more amino acid modifications (i.e.,substitutions, insertions or deletions) into an IgG constant domain, orF_(c)R binding fragment thereof (such as an F_(e) or hinge F_(e) domainfragment), see, e.g., WO 98/23289; WO 97/34631; and U.S. Pat. No.6,277,375.

Further, an antibody can be conjugated to albumin to make an antibodymore stable in vivo or have a longer half life in vivo. The techniquesare known in the art, see e.g., WO 93/15199, WO 93/15200 and WO01/77137; and EPO 413, 622. The antibody also can be modified, forexample, by glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein and so on.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine or other “caine”anesthetic to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water-freeconcentrate in a sealed container, such as an ampule or sachetindicating the quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampule of sterile water forinjection or saline can be provided, for example, in a kit, so that theingredients may be mixed prior to administration.

The invention also provides that a liquid formulation of the presentinvention is packaged in a sealed container such as an ampule or sachetindicating the quantity of the product of interest. The liquidformulations of the instant invention can be in a sealed containerindicating the quantity and concentration of the antibody or antibodyfragment. The liquid formulation of the instant invention can besupplied in a sealed container with at least 15 mg/ml, 20 mg/ml, 30mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml, or 300 mg/ml of CXCR5 antibodyin a quantity of 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml,10 ml, 15 ml or 20 ml, for example.

An article of manufacture containing materials useful for the treatmentof the disorders described above is provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for diagnosing,preventing or treating a CXCR5 condition or disease and may have asterile access port (for example, the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The label on or associated with the containerindicates that the composition is used for treating the condition ofchoice. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including buffers, diluents, filters, needles, syringes andpackage inserts with instructions for use.

In another aspect of the invention, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of CXCR5, by way of gene therapy.Gene therapy refers to therapy performed by the administration to asubject of an expressed or expressible nucleic acid of interest. In theembodiment of the invention, the nucleic acids produce the encodedprotein in and by target host cells that mediate a therapeutic effect.Any of the methods for gene therapy available can be used according tothe instant invention.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488 (1993); Wu et al., Biotherapy 3:87 (1991);Tolstoshev, Ann Rev Pharmacol Toxicol 32:573 (1993); Mulligan, Science260:926 (1993); Morgan et al., Ann Rev Biochem 62:191 (1993); and May,TIBTECH 11:155 (1993).

In one aspect, the compound comprises nucleic acid sequences encoding anantibody, or functional binding fragments thereof, said nucleic acidsequences being part of expression vectors that express the antibody orfragments or chimeric proteins or heavy or light chains thereof in asuitable host. In particular, such nucleic acid sequences have promotersoperably linked to the antibody coding region, said promoter beinginducible or constitutive, and, optionally, tissue-specific, as well asother regulatory sequences.

In another particular embodiment, nucleic acid molecules are used inwhich the antibody coding sequences and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe antibody-encoding nucleic acids (Koller, et al., Proc Natl Acad SciUSA 86:8932 (1989); Zijlstra et al., Nature 342:435 (1989)). In specificembodiments, the expressed antibody molecule is a single chain antibody;alternatively, the nucleic acid sequences include sequences encodingboth the heavy and light chains, or fragments thereof, of the antibody.Alternative methods for integration include using particulartranscription factors that recognize specific nucleic acid sequences,zinc fingers and so on.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient.

In one embodiment, the nucleic acid sequences are directly administeredin vivo and is expressed to produce the encoded product. That can beaccomplished by any of numerous methods known in the art, e.g., byconstructing the antibody encoding sequences as part of an appropriatenucleic acid expression vector and administering same so that thevectors become intracellular, e.g., by infection using defective orattenuated retrovirals or other viral vectors (see U.S. Pat. No.4,980,286), by direct injection of naked DNA, by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), using non-viralvectors, such as synthetic compositions comprising an amphipathiccompound that binds the hydrophilic nucleic acid and has the ability tofuse with cells, generally thus containing a hydrophobic portion forcombining with membranes, coating with lipids or cell-surface receptorsor transfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, by administering the vector in linkage to a peptide whichis known to enter the nucleus, by administering the vector in linkage toa ligand subject to receptor-mediated endocytosis (see, e.g., Wu et al.,J Biol Chem 262:4429 (1987)) (which can be used to target cell typesspecifically expressing the receptors) etc. In another embodiment,nucleic acid-ligand complexes can be formed in which the ligandcomprises a fusogenic viral peptide to disrupt endosomes, allowing thenucleic acid to avoid lysosomal degradation. In yet another embodiment,the nucleic acid can be targeted in vivo for cell-specific uptake andexpression, by targeting a specific receptor (see, e.g., WO 92/06180; WO92/22635; WO92/20316; WO93/14188 and WO 93/20221).

Regarding vectors, for example, a lentiviral vector can be used as knownin the art. The lentiviral vectors contain components for packaging theviral genome and integration into the host cell DNA. The nucleic acidsequences encoding the antibody to be used in gene therapy are clonedinto one or more vectors, which facilitate the delivery of the gene intoa patient. For example, a lentiviral vector can be used to deliver atransgene to hematopoietic stem cells. References illustrating the useof retroviral vectors in gene therapy are: Clowes et al., J Clin Invest93:644 (1994); Kiem et al., Blood 83:1467 (1994); Salmons et al., HumanGene Therapy 4:129 (1993); and Grossman et al., Curr Opin Gen and Dev3:110 (1993).

Adenoviruses also may be used in the instant invention. Targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells and muscle, for example. Adenoviruses infectnon-dividing cells, an advantage over early retroviral vectors. Kozarskyet al., Curr Opin Gen Dev 3:499 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431 (1991); Rosenfeld et al., Cell 68:143 (1992);Mastrangeli et al., J Clin Invest 91:225 (1993); WO94/12649; and Wang etal., Gene Therapy 2:775 (1995).

Adeno-associated virus (AAV) also can be used in gene therapy (Walsh etal., Proc Soc Exp Biol Med 204:289 (1993); and U.S. Pat. Nos. 5,436,146;6,632,670; and 6,642,051).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate-mediated transfection or viral infection. Usually, themethod of transfer includes the transfer of a selectable marker to thecells. The cells then are placed under selection to isolate those cellsthat have taken up and are expressing the transferred gene. Those cellsthen are delivered to a patient.

Thus, the nucleic acid can be introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion etc.Numerous techniques are known in the art for the introduction of foreigngenes into cells (see, e.g., Loeffler et al., Meth Enzymol 217:599(1993); Cohen et al., Meth Enzymol 217:618 (1993); and Cline Pharm Ther29:69 (1985)) and may be used in accordance with the present invention,provided that the necessary developmental and physiological functions ofthe recipient cells are not disrupted. The technique should provide forthe stable transfer of the nucleic acid to the cell, so that the nucleicacid is expressed by the cell, heritable and expressed by the cellprogeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include, but arenot limited to, epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes, blood cells, such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes and granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver etc.

In one embodiment, the cell used for gene therapy is autologous to thepatient. Nucleic acid sequences encoding an antibody of the instantinvention are introduced into the cells such that the transgene isexpressed by the cells or their progeny, and the recombinant cells thenare administered in vivo for therapeutic effect. In a specificembodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with the embodiment of the instantinvention (see e.g., WO 94/08598; Stemple et al., Cell 71:973 (1992);Rheinwald Meth Cell Bio 21A:229 (1980); and Pittelkow et al., MayoClinic Proc 61:771 (1986)). Because CXCR5 is expressed on, for example,B cells, blood cells and bone marrow cells are suitable host cells.However, the scope of the instant invention regarding the use of stemcell hosts does not contemplate the making and using of a transgene tomake a transgenic organism by administering the transgene of interest toembryos and embryonic stem cells.

The invention provides methods of treatment, prophylaxis andamelioration of CXCR5 diseases or one or more symptoms thereof byadministrating to a subject of an effective amount of, for example, aliquid formulation of the invention. The subject is preferably a mammalsuch as non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.)and a primate (e.g., monkey, such as a cynomolgus monkey, and a human).In a preferred embodiment, the subject is a human.

CXCR5 also is expressed on certain cancer cells, such as pancreas, colonand bladder, as well as on T cell leukemias (Qinping et al., Oncogene24:573-584, 2005), and B cell leukemias (Burkel et al., Blood, July2007; doi:10.1182/blood-2007-05-089409) and stimulation of CXCR5correlated with proliferation of carcinoma cells, Meijer et al., CancRes 66:9576-9582, 2006.

Thus, the antibody or derivative thereof of interest can be used tocontrol proliferation of cancer cells expressing CXCR5, which cancersare identified by determining presence of CXCR5 expression by adiagnostic assay taught herein. The antibody of interest can reduceinfiltration of malignant cells, reduce resistance to apoptosis andminimize proliferation. Such patients then are administered a cancercell proliferation inhibiting amount of an antibody, or derivativethereof, of interest as provided herein.

Autoimmune disorders are associated with aberrant and/or high expressionof CXCL13, such as lupus (Ishikawa et al., J Exp Med 193:1393-1402,2001) and Sjoren's Syndrome (Salomonsson et al., Scan J Imm 55:336-342,2002; and Barone et al., Arth Rheum 52(6)1773-1784, 2005), or highexpression of CXCR5, such as in myasthenia gravis (Sims et al., J Imm167:1935-1944, 2001; Saito et al., J Neuroimm 55:336-342, 2005; andTackenberg et al., Eur J Imm 37:849-863, 2007). Hence, an antibody ofinterest is used to minimize the effect of high levels or high activityof CXCL13 or CXCR5 ligand. In autoimmune disorders characterized by highlevels of B cells, high levels of CXCR5 or high levels of CXCL13, orother CXCR5 ligand, a B cell activity inhibiting amount of an antibodyof interest is administered as taught herein.

Aberrant CXCR5 expression is observed in multiple sclerosis, Brain129(Pt 1)200-211, 2006.

In colitis, CXCR5 has a role in GALT formation and function (Carlsen etal., Gut, 2002, 51(3)364-367). CXCR5-mediated migration and infiltrationof B cells into the gut lamina propria, and mucosal infiltration ingeneral, (Mazzucchelli et al., J Clin Invest, 1999, 104(10)R49-R54) andexpression thereof in ulcerative colitis lesions which contain ectopicgerminal centers, is inhibited by an antibody of interest.

B cell depletion can be of therapeutic benefit in ameliorating symptomsunder certain circumstances and in certain indications, such as, inrheumatoid arthritis (Oligino & Dalrymple, Arth Res Ther 5(Suppl4)S7-S11, 2003). CXCR5 is expressed at high levels in the synovialtissue of arthritis patients, as compared to tissue from individuals notafflicted with rheumatoid arthritis (Schmutz et al., Arth Res Ther7:R217-R229, 2005). Thus, certain forms of the instant antibody, andderivatives thereof, can deplete B cell populations, and can preventinfiltration and interaction of B cells in a joint. Accordingly, atreatment can include administering a B cell level reducing amount of anantibody of interest to a patient diagnosed with arthritis. The antibodycan be administered locally to the affected joint.

Ectopic lymphoid neogenesis is observed in several conditions, includingpsoriatic arthritis (Canete et al., Ann Rheum Dis, Jan. 12, 2007,doi:10:1136/ard.2006.062042), chronic inflammatory diseases, in general(Aloisi & Pujol-Borrell, Nat Rev Imm 6:205-217, 2006) and in graftsundergoing rejection, both chronic (Baddoura et al., Am J Trans5:510-516, 2005) and acute (DiCarlo et al., Am J Trans 7:201-210, 2007).CXCL13 and CXCR5 were present in cardiac grafts (DiCarlo et al., supra);and CXCL13 was present in psoriatic arthritis (Canete et al., supra).Presence of CXCL13 and/or CXCR5 is associated with the development ofthe ectopic lymphoid follicles with B cell and T cell areas, as found innormal nodes. B cell antigen presentation of alloantigen also wasassociated with acute cardiac allograft model (Noorchashm et al., J Imm177:7715-7722, 2006).

Thus, an antibody of interest can be used to dampen inflammation andgraft rejection. A patient then is administered a B cell activityinhibiting amount of an antibody to dampen inflammation, to minimizeectopic germinal center development, to minimize B cell recruitment to agraft and to minimize B cell alloantigen presentation before orfollowing a transplantation procedure.

The invention now will be exemplified for the benefit of the artisan bythe following non-limiting examples that depict some of the embodimentsby and in which the instant invention can be practiced.

EXAMPLES Example 1 Generation of Immunogen

Anti-CXCR5 monoclonal antibodies can be raised to CHO cells transformedwith DNA encoding full-length human CXCR5 and expressed on the cellsurface (“r-CXCR5-CHO cells”). The CXCR5 sequence used to transform thecells can be obtained from data bases, such as NM 001716.2, NC000011.8or NM001716, and synthesized or isolated from a suitable cell source.

The CXCR5 open reading frame was placed into an expression vector, suchas pcDNA3.1neo_DEST, and then transfected into 300-19 cells (Immunogen).

Also, the CXCR5 EC domain, with the amino acid sequence,MNYPTLEMDLENLEDLFWELDRLDNYNTSLVENHLC (SEQ ID NO:1), was conjugated toKLH by the C terminal cysteine, and was used as immunogen. Cellsexpressing CXCR5 or the CXCR5 EC domain were administered IP (5×10⁶cells in 0.2 ml or 50 μg peptide in 100 μl of buffer, optionally mixedwith 100 μl of adjuvant, such as Freund's complete adjuvant). Injectionswith the antigen were repeated every two weeks until high titer CXCR5antibody was detected in the serum using any of a variety of knownmethods, for example, by ELISA or FACS using, for example, CXCR5⁺ cells,which can be isolated, for example by FACS, and, for example, thecommercially available MAB 190 (R & D Systems) as a positive control.

The cells expressing CXCR5 were maintained at 37° C. under 5% CO₂ inRPMI (Invitrogen, Carlsbad, Calif.) supplemented with 10% dialyzed fetalbovine scrum (FBS) (Invitrogen). Cells were prepared for injection bysubstituting the above culture medium with phosphate-buffered(Ca/Mg-free) saline (CMF-PBS) supplemented with 5 mM EDTA, andharvesting the cells in that buffer. The harvested cells were pelletedby centrifugation at 500×g for about 5 minutes, washed once byresuspending the pellet in CMF-PBS and centrifuging as before, counted,and adjusted to the appropriate volume (such as 5×10⁶ cells in 0.2 ml)for injection by resuspending the cell pellet in CMF-PBS.

As mentioned, CXCR5 expression was monitored, for example, by FACSanalysis, using commercially available CXCR5 antibodies, such as MAB190(R & D), clone RF8B2 and 2G8 (2G8 is an rat anti-mouse CXCR5 antibody,while the other purchased antibodies are anti-human CXCR5 antibodies)(BD), and 2C1 (Abnova), as well as various polyclonal antibodiesdirected to hCXCR5 made practicing methods known in the art.

To facilitate the plasmid construction and to enhance the expression ofCXCR5, oligonucleotides corresponding to the leader peptide sequencecomprising the first 135 base pairs of the CXCR5 nucleic acid codingsequence were generated. The oligonucleotides contained some changes inthe wobble coding positions to lower the GC content. All nucleotidesequence changes were silent, i.e., no amino acid sequence changesresulted. After annealing the oligonucleotides together, the engineeredleader peptide coding sequence was linked to the rest of the codingsequence by PCR-SOE (Ho et al., Gene 77:51 (1989); and Horton et al.,BioTechniques 8:528 (1990)).

Expression of CXCR5 was verified prior to use as immunogen. Cells arecultured in RPMI (Invitrogen, Carlsbad, Calif.) containing 10% FBS, 0.2mM of glutamine and 1× non-essential amino acid solution followed byseeding about 3−5×10⁵ cells per well in a T75 flask and grown forapproximately 24-48 hours.

The transformed or transfected cells were cultured for about two weeksuntil the cells not carrying CXCR5 expression plasmid were eliminated byantibiotic selection. Cells of the stable cell lines can be lysed,proteins obtained and subjected to Western blot analysis.

Stable or transient transfected cells were assayed for expression ofCXCR5 using methods for detecting cell surface expression of CXCR5, suchas by FACS analysis. Alternatively, cells can be lysed and the proteinsstudies, for example, by Western blot analysis. Transfected cellsharvested from culture dishes were washed once with phosphate-bufferedsaline (PBS) and resuspended in deionized water, mixed with an equalvolume of 2× protein sample loading buffer (BioRad, Hercules, Calif.)and then heated at about 100° C. for 10 minutes. Membrane protein wasanalyzed using conditioned medium mixed with an equal volume of 2×protein sample loading buffer and heated at 100° C. for 10 minutes. Thesamples were separated using 4-12% gradient SDS-PAGE. The proteins weretransferred from the gel to a nitrocellulose membrane (BioRad, Hercules,Calif.), which was blocked with 5% nonfat dry milk in PBST (PBS with0.05% TWEEN-20®) for at least one hour prior to transfer of protein.

CXCR5 was detected by incubating the membrane with CXCR5-specificprimary antibody in blocking buffer for at least an hour at roomtemperature, with shaking. The membrane was washed at least three timesand a reporter-conjugated secondary antibody in blocking buffer wasadded to the membrane and incubated for at least one hour at roomtemperature, with shaking. The membrane was washed three times in PBSTand developed with, for example, a chemiluminescent substrate.

Example 2 Generation of Anti-CXCR5 Mabs

A/J or BALB/cJ mice, about 4-6 weeks old (Jackson Labs, Bar Harbor, Me.)were immunized with the CXCR5-transfected cells or an EC peptide. Agroup of mice were primed intraperitoneally on day 0 with a 1:1 emulsionof KLH-conjugated peptide mixed with adjuvant (CFA), boosted ip on day20 with the peptides with IFC (incomplete Freund's adjuvant) and/orcells in PBS without adjuvant, and finally boosted intravenously on day44 with the KLH-peptides mixed in IFC and/or cells in PBS, withoutadjuvant. Another group of mice were primed ip on day 0, boosted ip ondays 15, 39, 53 and 67, and finally boosted intravenously on day 81 (allinjections with cells in PBS, without adjuvant). For both groups ofmice, each injection contained approximately 3×10⁶ to 2×10⁷ cells in avolume of approximately 200 μl. Alternatively, peptide and/or cellimmunizations were performed once every two weeks, 3-6 times until adesirable anti-CXCR5 titer was obtained, as ascertained, for example, byFACS analysis or ELISA.

Three days after the last injection, the mice, optionally were testedfor anti-CXCR5 antibody titer in serum, were sacrificed and the spleenwas removed and placed in approximately 10 ml of serum-free DMEM (Gibco)in a Petri dish. The splenocytes were teased out of the capsule usingforceps and washed twice in 10 ml of serum-free IMDM (Cellgro, Herndon,Va.) at 37° C. The spleen cell suspensions were transferred to a 15 mlconical bottom tube and debris allowed to settle for about 2-5 minutes.The supernatant containing the splenocytes was transferred to a fresh 15ml conical bottom tube and washed three more times with IMDM until thefusion. The spleen cells from mice can be pooled.

Optionally, a 5 ml single cell suspension of control spleen feeder cellswas prepared from an unimmunized mouse essentially as described abovefor the immunized spleen cells and placed in an incubator (37° C., 5%CO₂) until needed.

The fusion partner for the immunized spleen cells can be ahypoxanthine/aminopterin/thymidine (HAT)-sensitive, non-secretingmyeloma cell line, such as P3×63-AG8.653 or SP2/0 (ATCC, Manassas, Va.)or FO_B lymphoblasts (ATCC, CRL-1646)). Prior to the fusions, thelymphoid cells were maintained in IMDM/10% FBS (37° C., 7% CO₂) ensuringthat the cells are in logarithmic growth phase on the day of the fusion.An alternative selection mechanism relies on using azaserine, whichtypically is added one day after fusion.

The fusion protocol used is a hybrid of the protocols set forth inLerner (Yale J Biol Med, 1981, 54(5)387-402) and Gefter et al. (SomaticCell Genet, 1977, 3(2)231-236). Before the fusion, the pooled spleencells were washed three times with serum-free IMDM, and counted. Also,immediately before fusion, the logarithmic phase mycloma cells werewashed three times with serum-free IMDM and counted. The lymphoid cellswere resuspended to 1×10⁷ cells/ml in serum-free IMDM. For each fusion,1−1.5×10⁸ spleen cells were mixed with 1-3×10⁷ myeloma cells in a 50 mlconical bottom polypropylene tube, and the cells were washed once withserum-free IMDM. The ratio of spleen cells to myeloma cells was 5:1. Thetubes were centrifuged at 500×g for 10 minutes to pellet the cells.After aspiration of the supernatants, the pellets were resuspendedgently by tapping the bottom of the tubes. The tubes then were placed ina beaker of 37° C. water. All subsequent fusion steps were carried outin that beaker.

Next, 1 ml of polyethylene glycol 1500 (Roche Applied Science,Indianapolis, Ind.) preheated to 37° C. was added slowly to each cellpellet over the course of about 1 minute, while gently rocking the tube.The cells were incubated in the PEG for about one minute followed byaddition of one ml serum-free IMDM added dropwise to each pellet overthe course of 30 seconds, and then 9 ml serum-free IMDM were added toeach pellet over the next minute. Both tubes were centrifuged at 500×gfor 10 minutes at room temperature, and the supernatants were aspirated.The cell pellet was resuspended in 100 ml of filtered complete hybridomaproduction media (500 ml IMDM (Cellgro) mixed with 10% FBS (SeraCare,Millford, Mass.), 0.2 mM of L-glutamine, 1× non-essential amino acidsolution, 1 mM sodium pyruvate, 0.01% pen-strep (Invitrogen) and 1× HTsupplement (Invitrogen)).

Each 100 ml cell suspension was plated in ten 96-well flat-bottommicrotiter plates, with a volume of ˜100 μl/well. The plates were keptin an incubator at 37° C., 7% CO₂. On day 2 post-fusion, the cells wereselected by addition of 5.7 μM azaserine in IMDM to the fused cells at100 μl per well. Supernatants were withdrawn for primary screening,typically on days 10-14 post-fusion, from wells containing clones. Thefusion efficiency was 75-99% (720-950 out of 960 possible wellsdeveloped clones that were screened).

The primary screen can be a radioimmunoassay (RIA) designed to detectantibodies that bind to human CXCR5. To perform the RIA,affinity-purified goat anti-mouse IgG (F_(c) fragment-specific)(Cappell, Cochranville, Pa.) in PBS is added to 96-well PVC microtiterplates (50 μl/well) and incubated overnight at 4° C. The goat anti-mouseIgG is removed from the plates and the wells are blocked with 100μl/well of 5% FCS/PBS for 1 hour at room temperature. After removing theblocking solution, neat hybridoma culture supernatant is added to thewells (50 μl/well) and incubated 1 hour at room temperature. The platesare washed 3 times with PBS/0.05% Tween-20. Next, 50 μl ¹²⁵I-CXCR5(˜20,000 cpm) in PBS/5% FCS are added to each well, and incubated 1 hourat room temperature. Finally, the wells are washed 3 times withPBS/0.05% Tween-20. After flicking out all the wash buffer, the wellsare separated by cutting the plates and analyzed in a gamma counter.Wells to which 5% FCS/PBS is added instead of culture supernatant servedas background wells.

The purified CXCR5 is labeled with ¹²⁵I according to the Bolton-Huntermethod, substantially as described by the supplier of the Bolton-Hunterreagent (New England Nuclear, Boston, Mass.). The quality of the¹²⁵I-CXCR5 is monitored by confirming that the labeling procedure didnot destroy the epitopes recognized by commercially available CXCR5antibodies (R & D or Becton Dickinson, Mountain View, Calif.).

Clones are considered to be positive on primary screening if supernatantsamples are labeled approximately 10-fold over background in the RIA.Positive clones are pulled, expanded and stored frozen.

A primary hybridoma screen was designed to determine whether theantibodies recognized native CXCR5 epitopes. That was accomplished byFACS analysis of cell surface CXCR5 displayed on CXCR5⁺ cells stainedwith the monoclonal antibodies, visualizing binding with fluorescentlylabeled goat anti-mouse second antibody. Clones were considered to bepositive on primary screening if supernatant samples were labeledapproximately 10-fold over background in the FACS analysis. Also, tolocalize the CXCR5 epitopes bound by antibodies, competition assays withCXCR5 antibodies were conducted. Positive clones were selected, expandedand stored frozen.

Example 3 Cell-Based Binding Assays for Anti-CXCR5 mABS

A cell-based binding assay was used to characterize the anti-CXCR5 mAbs.For example, the CXCR5-expressing transfected cells described above,such as hCXCR5/HEK293, can be used. A full-length human CXCR5 openreading frame was cloned into a vector, for example, pcDNA3.1neo DEST(Invitrogen, Carlsbad, Calif.). The CXCR5-coding region was synthesizedby RT-PCR using human brain and liver RNA (Ambion, Inc., Austin, Tex.)as a template. The final plasmid construct, CXCR5/cDNA3.1neo, expresseda full-length CXCR5 protein. A stable cell line expressing CXCR5 wasgenerated by transfection of CXCR5/pcDNA3.1neo plasmid construct intoCHO or HEK293 cells (ATCC No. CRL-1573) using a standard andcommercially available Lipofectamine 2000 kit. After transfection, thecells were cultured in DMEM overnight, then reseeded in medium with 200μg/ml neomycin and cultured for 12-14 days. Isolated single colonieswere picked and grown in separate wells until enough clonal cells wereamplified. Stable clones resistant to neomycin and which expressed highlevels of CXCR5 protein were identified by FACS analysis usingpolyclonal anti-CXCR5 antibodies (R&D Systems, Minneapolis, Minn.) orcustom generated polyclonal antibodies.

Human HS Sultan cells (ATCC No. CRL-1484) naturally expressing CXCR5were also confirmed for CXCR5 expression by FACS analysis. HS Sultancells were grown in RPMI 1640 containing 10% fetal calf serum, 0.2 mM ofglutamine and 0.1% pen/strep solution (100 μg/ml penicillin and 10 μg/mlstreptomycin).

Cell-based antibody-binding can be assessed using the FMAT™(fluorescence macro-confocal high-throughput screening) 8100 HTS or 8200Cellular Detection System (Applied Biosystems, Foster City, Calif.)following the protocol provided by the manufacturer. Cell linesnaturally expressing CXCR5 or stably transfected with CXCR5 expressionconstructs are seeded in 96-well plates. Alternatively, transientlytransfected 293T or CHO cells are seeded in the 96-well plate. The cellsare seeded at a density of 5,000-30,000 cells per well. After 20-24hours, anti-CXCR5 mAbs and FMAT-conjugated goat anti-mouse IgG antibodyare added together to the wells and incubated for 1 h, 2 h, 4 hr orovernight at room temperature.

Cell-based antibody-binding was also assessed by FACS using aHEK293/CXCR5 stable cell line expressing CXCR5. Cells were incubatedwith anti-CXCR5 mAbs in PBS. After three washes, the cells wereincubated with fluorescent molecule-conjugated secondary antibody (BDSciences, Palo Alto, Calif.).

The results indicated that several mAbs bind to CXCR5 expressed fromeither recombinant plasmid constructs. For example, clones 11D6, 14C9,19H5, H28, 54G6, G7, 56H6, 79B7 and 16D7, as well as humanized variantsof the latter antibody, 16D7, 16D7-HC1-LC3, 16D7-HC1-LC2, 16D7-HC1-LC1and 16D7-HC2-LC1, a negative control IL13 antibody, CA13, positivecontrols, MAB190, 2C1 and RF8B2, three mouse isotype controls to IgG1,IgG2a and IgG2b and a rat IgG2b isotype control (matched to RF8B2), weretested for binding to HEK293 cells transfected to express CXCR5. The 2C1and MAB 190 positive control antibodies bind to the CXCR5 cells. RF8B2presented with intermediate binding levels. The negative controlantibodies exhibited only background binding. All antibodies except forCA13 bound to the hCXCR5/HEK293 cells with similar binding profiles andtitration kinetics as the parent antibody 16D7 and with 79B7.

Transiently transfected HEK293 cells containing a CXCR5/neo plasmid arealso stained with immunofluorescence as described above and observed byfluorescent microscopy. The cell-based FMAT and FACS analyses confirmthat mAbs indeed bind to CXCR5 expressed either from recombinant plasmidconstructs or as native protein in cultured cells. A positive bindingsignal is determined based on the FMAT signal read-out that issignificantly higher than background binding and other negativehybridoma clones (p>0.01).

The CXCR5 mAbs generated, such as 16D7, 14C9, 19H5, H28, 54G6, G7, 56H6and 79B7, bind to the EC domain and block binding of CXCL13 to CXCR5 onthe cell.

Example 4 Biacore Affinity Analysis

The N-terminal EC region of CXCR5 (amino acids 1-59) from human andmouse were synthesized with a terminal biotin tag, and used in a forwardformat Biacore assay where the peptides were immobilized on a Biacorechip and then the kinetics of antibody interaction with the peptides onthe chip were determined. The synthetic peptides were immobilized on aBiacore chip for approximately 20 response units (RUs'). Then the mAb'swere exposed to the chip for kinetic measurements, following themanufacturer's recommendations (GE Healthcare, Piscataway, N.J.).

Mouse anti-hCXCR5 mAb clone, 16D7, had a calculated K_(D) of 2.16⁻¹² M;mouse/human IgG4 chimeric 16D7 (16D7 VH and VL regions grafted onto ahuman IgG4 Fc, the sequence of which is known in the art, optionallycodon optimized, using standard methods, such as cloning, amplificationof ends, ascertaining the mass of the regions and cloning the portions)had a K_(D) of 1.41⁻¹² M; and for various humanized variants of 16D7,wherein the structure of the variants and the derivation of the heavyand light chains thereof is denoted by the terms, “HC_” for a particularheavy chain and “LC_” for a particular light chain, which are graftedonto an IgG4 backbone, where the composition of the chains is providedhereinbelow, 16D7-HC1-LC1 had a K_(D) of 3.11⁻¹² M; 16D7-HC1-LC2 had aK_(D) of 1.41⁻¹² M; 16D7-HC2-LC1 had a K_(D) of 2.40⁻¹² M; 16D7-HC1-LC3had a K_(D) of 1.21⁻¹² M; 16D7-HC3-LC4 had a K_(D) of 4.92⁻¹² M;16D7-HC3-LC5 had a K_(D) of 1.84⁻¹⁰ M; and 16D7-HC1-LC6 had a K_(D) of9.17⁻¹¹ M.

Humanized SAR113244, a form of the 16D7 humanized variant, 16D7-HC1-LC3that carries the S241P and L248E substitutions (substitutions introducedpracticing known methods and reagents, using Kabat numbering), wascaptured on a Biacore chip by pre-immobilized mouse anti-human IgG Fcantibody, and then used in a reverse assay format Biacore assay wherethe kinetics of the un-tagged human CXCR5 N-terminal peptide (aminoacids 1-59) interaction with the mAb's on the chip were determined. TheK_(D) for SAR113244 was determined to be 1.13±0.08⁻¹¹ M. The K_(D)values determined with the forward assay using biotinylated humanpeptide immobilized on the chip surface and SAR113244 as analyte, wereconsistent with those obtained using the reverse assay.

Example 5 Western Blot Analysis of Anti-CXCR5 mABS Binding Activity

Western blot was performed to assess the anti-CXCR5 mAb binding activityto CXCR5 under denaturing condition, as well as, expression levels ofCXCR5 and other CXCR5-related protein in human cell lines. Proteinsamples also were prepared from stably transfected cells using M-PERmammalian protein extraction reagent kit (Pierce, Rockland, Ill., Cat#78501) following manufacturer's instructions, and heated at 70° C. for10 minutes after adding an equal volume of 2× protein sample loadingbuffer. All samples were separated by electrophoresis in a 4-12%gradient SDS-PAGE gel. The proteins were transferred from the gel to aPVDF membrane and anti-CXCR5 mAbs were applied to the Western blotmembrane as the primary detection antibody. An Alexa 680-conjugatedsecondary antibody was used for detection and the membranes were scannedusing the Odyssey Infrared Imaging system (Li-cor, Lincoln, Nebr.) orusing electrochemiluminescence (ECL). Positive control antibodiesagainst human CXCR5 were generated as taught herein.

Example 6 FACS Assay for Monitoring CXCR5 Internalization

Buffy coat cells are obtained from healthy volunteers (Gulf Coast BloodCenter, Houston, Tex.). Human peripheral mononuclear cells (PBMCs) areisolated with a standard Ficoll-Hypaque gradient method. PBMCs arecultured (0.5×10⁶ cells/well) in 96-well plate at 4° C. Each wellcontains 0.2 ml of RPMI 1640 supplemented with 10% FBS in thepresence/absence of monoclonal antibodies (10 μg/ml). After 30 minutes,the medium is replaced with fresh, cold RPMI 1640 supplemented with 10%FBS and no antibodies. The cells are transferred to a 37° C. humidifiedtissue culture chamber containing 5% CO₂. Monoclonal antibody-treatedcells are harvested immediately, 2 hr or 24 hr after transferring thecells to 37° C. Cells are washed once with PBS and incubated in cold PBScontaining 1% BSA (PBSB) for 30 minutes. Cells then are stained withPE-conjugated anti-human CXCR5 (BD Biosciences). After 30 minutes, cellsare washed 3 times with PBSB and fixed in 1% paraformaldehyde solutionovernight. The next day, presence of CXCR5 is analyzed with a BDFACSCalibur™ system flow cytometer (BD Biosciences, San Jose, Calif.).

Example 7 FLIPR Assay

Changes in intracellular calcium were measured by plating 9000cells/well and incubating overnight. The cells were the RBL-2H3 linestably transfected with human CXCR5. Cells then were washed and thenloaded with 2 mM fluo-4/AM (Molecular Probes) in a buffer containing 2.5mM probenicid. Cells were exposed to CXCR5 mAb then washed with assaybuffer. The cells were exposed to 10 nM CXCL13 (R & D). Changes inintracellular Ca⁺² were recorded using the 384-B FLIPR device (MolecularDevices). Commercially available anti-human CXCR5 mAbs, and mouse IgG1and IgG2b were used as controls.

As discussed herein below, several humanized versions of mAb 16D7 wereconstructed, such as chimeric 16D7 (the hIgG₄ chimera), 16D7-HC1-LC1,16D7-HC1-LC2, 16D7-HC2-LC1, 16D7-HC1-LC3, 16D7-HC3-LC4, 16D7-HC3-LC5 and16D7-HC1-LC6, and they were tested for biological activity as evidencedby calcium flux.

The humanized antibodies, aside from a negative control, CA13,demonstrated signal neutralizing activity equal to that of the parent16D7 antibody on transfected cells stably expressing CXCR5.

Example 8 Chemotaxis Assay

CXCR5⁺ HS Sultan cells (ATCC CRL1484) were added to the upper chamber ofa transwell plate (Millipore) at 0.5×10⁶ cells/well in the presence of100 nM CXCL13 (R & D) or CTX buffer (RPMI without phenol red, containing1% FBS, 0.5% BSA and 1 nM Na pyruvate) and migrating cells to the lowerchamber were assessed. The two chambers were assembled and incubated fortwo hours. Cells in the lower chamber were counted after addingcolorimetric reagent (Promega) and reading at OD₄₉₀.

CXCR5 specific migration was determined as the difference between thetotal number of migrated cells and the number of spontaneously migratingcells. If an anti-CXCR5 is tested, the cells are incubated with theantibody for 30 minutes prior to adding to the upper chamber. The degreeof antibody inhibition is the ratio of the specific migration in thepresence of antibody to the amount of migration in the absence ofantibody. That ratio can be multiplied by 100% to yield a percentinhibition metric.

The antibodies of Example 7 were compared to the parent 16D7 antibodyfor the ability to neutralize chemotaxis. All of the humanizedantibodies aside from negative control mAb CA13, neutralized chemotaxisin a profile comparable to that of 16D7 and 79B7, 14C9, 19H5, H28, 54G6,G7, 56H6. R&D MAB190 had intermediate activity while H28 and Abnovaantibody 2C1 did not completely neutralize ligand-induced cellmigration.

Example 9 Primary Human B Cell Reactivity Assay

Human PBMCs were isolated from whole blood using Accuspin columns(Sigma). PBMCs then were resuspended in BD Stain buffer (BectonDickinson) at 20 million cells/ml. One μg of mouse anti-human CXCR5monoclonal antibody was added to 50 μl PBMCs and allowed to bind for 20minutes at 4° C. Cells were washed two times with BD Stain buffer. Fiftyμl of the second antibody, goat-anti-mouse IgG-PE F_((ab′)) (BeckmanCoulter) diluted 1/100, were added to the PBMC-antibody cocktail andallowed to bind for 20 minutes at 4° C. Cells were washed three timeswith BD Stain buffer. A cocktail containing mouse anti-human CD20-FITC(BD) and CD4-APC (BD) at 1/50 dilution, each was added to the cells,which then were incubated for 20 minutes at 4° C. to assess B/T cellspecificity. Cells were washed 3 times with BD Stain buffer andresuspended in 250 μl BD Stain buffer and subjected to FACS analysis ona FACStar Plus. Mouse anti-human CXCR5 antibody (R&D; mAb190) is used asa positive control. Titration curves were generated for humanizedantibodies and Mean Fluorescence Intensity (MFI) plotted againstconcentration.

The humanized antibodies of Example 7 were tested for binding to humanPMBCs.

The antibodies, aside from negative control CA13, bind and have the sametitration profile on human B cells. Negative control CA13, demonstratedonly background binding. BD clone RF8B2 binds poorly to human PBMCs.

Example 10 Cynomolgus B Cell Reactivity Assay

Cynomolgus (cyno) monkey whole blood was obtained from Bioreclamation,Inc. (Hicksville, N.Y.). The blood was shipped in BD Cell PreparationTubes (CPT) post-centrifugation. The cyno PBMCs contained in the plasmalayer were removed from the CPT tube into a 50 ml tube leaving thegradient gel layer undisturbed. The tube was washed with 5 ml PBS tocompletely extract all the cells and the wash was added to a fresh 50 mltube. The cyno PBMCs were centrifuged at 1200 RPM for 10 minutes at 4°C. The pellet was resuspended in 1 ml BD FACS Stain buffer (BD). Onemillion cells were used per assay. One μg of mouse anti-human CXCR5monoclonal antibody (purified) was added to 50 μl PBMC and allowed tobind for 20 minutes at 4° C. Cells were washed 2 times with BD Stainbuffer. Fifty μl of second antibody, goat anti-mouse IgG-PE F_((ab′))(Beckman Coulter) diluted 1/100, was added to the cells and allowed tobind for 20 minutes at 4° C. Cells were washed three times with BD Stainbuffer. A cocktail containing mouse anti-human CD20-FITC (BD) andCD4-APC (BD) at 1/20 dilution each was added to cells for a 20 minuteincubation at 4° C. for assessment of B/T cell specificity. Cells werewashed 3 times with BD Stain buffer, and resuspended in 250 μl BD Stainbuffer and subjected to FACS analysis on a FACStarPlus. Commercial mouseanti-human CXCR5 mAb (R&D; MAB 190) was used as a positive control.

Mouse monoclonal 11D6 of the instant invention reactive to human CXCR5,was compared to an IgG isotype control. The humanized versions of 16D7and the commercially available MAB190 were tested for reactivity tocynomolgus CXCR5. 79B7 also was tested.

Cells positive for CD20 and CXCR5 were found with MAB190 and 11D6. Onthe other hand, 16D7 and the humanized variants thereof, as well as G7and BD RF8B2 and Abnova 2C1 did not bind to cynomolgus B cells. 14C9,19H5, H28, 54G6, 56H6 and 79B7 also bound cynomolgus B cells.

The instant CXCR5 antibodies were used to study peripheral blood cells.B cells expressed CXCR5 and in at least one experiment, about 10% ofperipheral T cells were found to express CXCR5.

Example 11 Sequencing of Anti-CXCR5 mABS

The mouse monoclonal antibodies were isotyped using a commerciallyavailable isotyping kit. The variable sequences of 16D7 and otheranti-CXCR5 mAb were sequenced. Total RNA was isolated from about 5million cells of the hybridoma using the Qiagen Qianeasy miniprep kit byfollowing the kit protocol. First strand cDNA was synthesized using theInvitrogen Superscript kit (Cat 11904-018), the kit protocols werefollowed.

The heavy chain and light chain variable regions were first amplifiedusing the following degenerate PCR primers and Taq polymerase (Roche)based on methods described in Wang et al. J Immunol Methods. 233:167-77,2000.

Heavy chain: Left primer: (SEQ ID NO: 2)1: CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC (SEQ ID NO: 3)2: CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG Heavy chain: Right primer:(SEQ ID NO: 4) GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC Light chain:Left primer: (SEQ ID NO: 5) GGAGCTCGAYATTGTGMTSACMCARWCTMCA Light chain:Right primer: (SEQ ID NO: 6)TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC

where R is either A or G; N is either A, G, T or C; M is either A or C;W is either A or T; S is G or C; and Y is C or T.

The PCR products were cloned into the pCR4-TOPO® using the InvitrogenTOPO TA Cloning® kit (Cat #: 45-0641) and sequenced using T3 and T7primers. Sequences then were blasted against the gene bank database todeduce the leader sequences for cloning of full variable regions. Basedon the blast results, the following primers (Chardes et al., FEBSLetters 452:386-394, 1999) were chosen for a second round of PCRamplification using Pfx polymerase (Invitrogen).

Heavy chain: Left primer: (SEQ ID NO: 7) CCAAGCTGTGTCCTRTCCRight primer: (SEQ ID NO: 8) CGACAAGTCGACTAGCCCTTGACCAGGCATCCLight chain: Left primer: (SEQ ID NO: 9) WTCTCTRGAGTCAGTGGGRight primer: (SEQ ID NO: 10) CGACTAGTCGACTGGTGGGAAGATGGATACAG

The PCR products were cloned into pCR-Blunt II®-TOPO® using theInvitrogen Zero Blunt® TOPO® PCR cloning kit (Cat 45-0245) and sequencedusing T7 primers.

Once the light and heavy chains are sequenced, the nucleic acids can berecoded to optimize expression in, for example, human host cells.

Example 12 Transfectomas

NS0-eu cells are grown to a density of 1×10⁶ cells/ml. The cells aremaintained in exponential growth phase and medium is changed the daybefore transfection. The day of transfection, 40×10⁶ cells are washed.Then, 10 μg of linearized nucleic acid, such as light chain DNA, and 10μg of, for example, linearized heavy chain DNA are added to the cellsuspension (the total DNA volume should be less than 50 μl) and theculture incubated on ice for 15 min. The DNA and cell mixture istransferred to a chilled cuvette (0.4 cm) and an electric pulse (750 Vand 25 μF) is applied. The cuvette is placed on ice immediately afterthe electric pulse and kept on ice for 10-15 min. The cells arecollected and plated. The cells are incubated in a 5% CO₂ incubator for12-16 days or until colonies appear. The supernatant of the cellcolonies or cells grown in suspension culture is tested by ELISA andpositive transfectomas are cloned in fresh medium. To further screen thepositive transfectomas, either titration ELISA or the Biacore assay isconducted. Expanded transfectomas are maintained in shaker flasks andantibody or derivative thereof collected from the supernatant.

Example 13 In Vivo Assays

Collagen-induced arthritis (CIA), a well-established model for human RA,has been used to demonstrate the efficacy of antibodies to TNFα(Williams et al., PNAS 1992, 89:9784-9788), as well as, fusion proteinsof CTLA-4 and TNFα (Webb et al., Eur J. Immunol. 1996, 26:2320-2328; andWooley et al., J. Immunol. 1993, 151:6602-6607). A rat anti-mouse CXCR5monoclonal antibody, clone 1038, was profiled in a mouse model of CIA inwhich DBA/1J mice were immunized and boosted with chick collagen typeII. Disease severity (which was visually scored by measuring pawswelling/inflammation) was monitored twice weekly, while jointscollected at study termination were evaluated for changes ininflammation, pannus, cartilage destruction and bone erosion. Clone1038, when administered in a prophylactic dosing regimen, significantlyreduced both disease severity and joint pathology compared toisotype-treated CIA mice (repeated measure ANOVA, p<0.05).

An acute mouse model for assessing efficacy of 16D7-HC1-LC3 in in vivochemotaxis was employed. Briefly, C57/B16 mice (8-16 weeks of age)selectively expressing huCXCR5 on immunocytes, such as B cells, T cellsand neutrophils, were generated by traditional transgenic methods usinga CD11a promoter. The in vivo chemotaxis model is a neutrophil-drivenmodel. On intraperitoneal administration of 20 μg of huCXCL13 ligand(R&D), mouse neutrophils expressing the huCXCR5 receptor migrated to theperitoneal cavity in response to a huCXCL13 gradient. Peritoneal cavitywashes were used to recover cells 80 minutes post-intraperitonealadministration of huCXCL 13 and fluorocytometric analysis was used toquantify the number of huCXCR5-expressing neutrophils in 2 ml samples ofperitoneal lavages that specifically migrated into the peritoneal cavityin response to huCXCL13 instillation. Neutrophils were identified byphenotypic markers, such as Ly6G, CD19 and CD11b. Subcutaneousadministration of humanized anti-hCXCR5, 16D7-HC1LC3, at two differentdoses (7.5 μg or 15 μg) 24 hours prior to instillation of huCXCL13showed efficacy in reducing huCXCR5-expressing neutrophil migration tothe peritoneal cavity in response to huCXCL13 when compared to anisotype-treated control, the two CXCR5 antibody treated samplesdemonstrating essentially no statistically different levels ofneutrophils as compared to the isotype negative control level ofneutrophils. At 1.5 μg, the humanized CXCR5 antibody showed a low levelof neutrophil migration inhibition as compared to the higher doses ofCXCR5 antibody tested.

Example 14 Resurfacing

Resurfacing of the murine 16D7 clone followed the steps described inProc. Natl. Acad. Sci. USA (1994) 91:969 and in U.S. Pat. No. 5,639,641.

The V_(L) and V_(H) sequences of 16D7 were blasted against the ProteinData Bank (Nucleic Acids Research, 28:235-242 (2000) or one can accessthe Protein Data Bank (PDB) on the internet, which contains the 3Dcoordinates of biological macromolecules, and the ten light and heavychain amino acid sequences most similar to that of 16D7 were retrieved.The PDB identification codes are used for identifying the sequences.

The ten closest homologues for the variable light chain were 1MJU (J MolBiol 332:423-435, 2003), 1AE6 (Proteins 29:161-171, 1997), 1QYG(Pozharski et al., “Carving a Binding Site: Structural Study of anAnti-Cocaine Antibody” in “Complex with Three Cocaine Analogs”), 1 UZG(J Virol 79:1223, 2005), 1UB5 (Beuscher et al., “Structure and Dynamicsof Blue Fluorescent Antibody 19G2 at Blue and Violet FluorescentTemperatures”), 1RUR (Proc Natl Acad Sci USA 110:2247-2252, 2004, 1FPT(Nat Struct Biol 2:232-243, 1995), 1QFU (Nat Struct Biol 6:530-534,1999), 1NAK (Virology 315:159-173, 2003) and 1CGS (J Mol Biol236:247-274, 1994) (redundant sequences were removed) and the tenclosest homologues for the variable heavy chain are 1FNS (Nat StructBiol 7:881-884, 2000), 1OAK (Nat Struct Biol 5:189-194, 1998), 1VFB(Proc Natl Acad Sci 91:1089-1093, 1994), 1CIC Nature 348:254-257, 1990),1GIG (Acta Crystallogr D Biol Crystallogr 50:768-777, 1994), 1T4K (J MolBiol 343:1269-1280, 2004), 1A7P (Marks et al.), 1FE8 (J Biol Chem276:9985-9991, 2001), 1DL7 (J Exp Med 191:2101-2112, 2000) and 1YY8(Cancer Cell 7:301-311, 2005). The closest homologs for the light andheavy chain were 1MJU and 1FNS, respectively. Those two sequences wereused to build a homology model of the variable domains which wassubsequently energy-minimized by a conjugate gradient minimization ofatomic coordinate positions with the CHARMM22 force field (J Comput Chem(1983) 4, 187; J Comput Chem (1986) 7, 591) as implemented in the MOEsuite (Chemical Computing Group, Quebec, CA). The model was used tolocate the CDR regions and the framework residues. The solventaccessibility for each variable region residue of the ten closesthomologs for each antibody variable region was calculated and averagedin an Excel spreadsheet as implemented in a Scitegic protocol (Hill &Lewicki (2006) Statistics: Methods and Applications, Statsoft, Tulsa,Okla.). Positions with greater than a 30% average accessibility wereconsidered surface residues. Positions with average accessibilities ofbetween 25% and 30% were further considered depending on proximity tothe CDR loops.

The surface positions of the murine 16D7 variable region were comparedto the corresponding positions in the human antibody sequences. Onlythose residues which displayed an accessible surface area greater than30%, with a few residues displaying an accessible surface area greaterthan 25% and which were flanking solvent exposed residues, were retainedfor the search. Some conserved residues in all immunoglobulin sequenceswere included to improve convergence of the search. Only germ linesequences were retained for analysis of the hits. The human antibodyvariable region surface with the most identical surface residues, withspecial consideration given to positions that come within 5.0 Å of aCDR, was chosen to replace the murine 16D7 antibody variable regionsurface residues.

None of the sequences contains any known B-cell or T-cell epitope listedin the Immune Epitope database (IEDB, Immune Epitope Database andAnalysis Resource web site; PLoS Biol. 2005; 3(3):e91).

The original sequences of murine 16D7 variable domains are:

the light chain (CDRs are underlined): (SEQ ID NO: 11)DIVMTQAAPSVAVTPRESVSISCRSSKSLLHSSGKTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYP YTFGGGTKLE IK; and the heavy chain (CDRs are underlined)(SEQ ID NO: 12) QVQLKESGPGLVAPSQSLSITCTVSGFSLIDYGVNWIRQPPGKGLEWLGVIWGDGTTYYNSALKSRLSIRKDNSQSQVFLKMNSLQTDDTAMYYCARIVYWGQGTLVTVSA.

The retained set of surface residues for the search of the light chainincluded D1, V3, A7, P9, P15, R16, E17, S18, P45, G46, Q47, D65, R79,R82, E86, K108, E110 and K112.

The query for the light chain contains the set of surface residuesdefined above and the conserved amino acids, C23, W40, Q43, Y91, C93,F103, G104, G106 and T107, which were included for the convergence ofthe BLAST protocol. All the other amino acids could be any of the 20naturally amino acids. The BLAST search was done against the humangermline antibody sequence database compiled by the IMGT (InternationalImmunogenetics Information Systems website, Molec Immunol, 2004,40:647-659). The best scoring match was found to be X72482(protein_id=CAA51150.1) from which was derived the light chain, LC4. LC5and LC6 are two variants of VL4 (VL means variable light) that aresuggested to address potential problematic residues in the light chain:1 exposed methionine (M51) to be mutated to Leu (LC5 & LC6) and 1potential deamidation site (LC6) where the asparagine N53 is changed toa serine residue. In total, 3 versions are proposed for the variablelight chain which contain between 4 and 6 mutations when compared to theparent murine 16D7 clone. The corresponding mutations are given in thefollowing Table 1. Sequential and Kabat numbering are given.

The retained set of surface residues for the variable heavy chainincluded Q1, Q3, K5, S7, P9, L11, S15, Q16, S20, P41, G42, K43, S61,A62, K64, S65, R70, S74, Q75, Q86, T87, D88, Q103, L106, A111, A112 andK113 (sequential numbering). The invariant amino acids which wereincluded in the BLAST query for the convergence of the search were: C22,W36, 137, Q39, D89, Y93, C95, W101, G102, G104 and T105. The BLASTsearch was done against the human germline antibody sequence databasecompiled by IMGT. One version for the heavy chain (HC3) was retained.The two V_(H) domains of AF062266 (protein_id=AAC18304.1) and AY393082(protein_id=AAS86018.1), that showed the best matching score for the setof surface residues, exhibit equivalent similarity score and displayidentical surface residues. Consequently only a single sequence for theheavy chain was retained with ten mutations. The lower scoring sequencesthat have different surface residues were not retained as they have lesspolar residues suggestive of potential reduced solubility.

TABLE 1 Light Light Chain Chain Ver- Ver- Ver- (sequential (Kabat sion5sion6 sion7 numbering) numbering) (LC4) (LC5) (LC6) Ala7 Ala7 Ser SerSer Pro9 Pro9 Leu Leu Leu Arg16 Arg16 Gly Gly Gly Met56 Met51 Met LeuLeu Asn58 Asn53 Asn Asn Ser Arg82 Arg77 Lys Lys Lys In total 4 5 6 forVL mutations mutations mutations Heavy Heavy Chain Chain (sequential(Kabat numbering) numbering) (HC3) (HC3) (HC3) Lys5 Lys5 Gln Gln GlnGln16 Gln16 Glu Glu Glu Ser61 Ser61 Pro Pro Pro Ala62 Ala62 Ser Ser SerArg70 Arg70 Ser Ser Ser Gln75 Gln75 Lys Lys Lys Gln86 Gln83 Thr Thr ThrThr87 Thr84 Ala Ala Ala Asp88 Asp85 Ala Ala Ala Ala111 Ala113 Ser SerSer In total 10 10 10 for VH mutations mutations mutations

Three versions are proposed for the light chain (LC4, LC5 and LC6).Individual mutations introduced through the resurfacing of the variablechains are noted in lowercase and underlined and CDRs are underlined.Resurfaced sequences of the variable regions are listed below, theconstant domain (IgG4) is not included.

LC4: (SEQ ID NO: 13) DIVMTQsAlS VAVTPgESVS ISCRSSKSLL HSSGKTYLYWFLQRPGQSPQ LLIYRMSNLASGVPDRFSGS GSGTAFTLkI SRVEAEDVGVYYCMQHLEYP YTFGGGTKLE IK LC5: (SEQ ID NO: 14)DIVMTQsAlS VAVTPgESVS ISCRSSKSLL HSSGKTYLYWFLQRPGQSPQ LLIYRlSNLASGVPDRFSGS GSGTAFTLkI SRVEAEDVGVYYCMQHLEYP YTFGGGTKLE IK LC6: (SEQ ID NO: 15)DIVMTQsAlS VAVTPgESVS ISCRSSKSLLHSSGKTYLYWFLQRPGQSPQ LLIYRlSsnLASGVPDRFSGS GSGTAFTLkI SRVEAEDVGVYYCMQHLEYPYTFGGGTKLE IK

One version was proposed for the heavy chain (VH3) (VH means variableheavy). Mutations introduced through the resurfacing of the variablechain are in lowercase and underlined, and the CDRs are underlined. Theconstant domain sequence is not included.

HC3: (SEQ ID NO: 16) QVQLqESGPG LVAPSeSLSI TCTVSGFSLIDYGVNWIRQPPGKGLEWLGVIWGDGTTYYN psLKSRLSIs KDNSkSQVFL KMNSLtaaDTAMYYCARIVYWGQGTLVTVS s

Nucleotide sequences were generated by OE-PCR and cloned intoNheI/HindIII sites of the episomal expression vector pXL4214 (Durocheret al., NAR, 2002, 30(2), E9. Sequences are codon optimized forexpression in human cells. V_(L) was fused to IGKC (AAH93097). V_(H) wasfused to IGHG4 (AAH25985), lacking the C-terminal Lys (IGHG4ΔK).Sequences were validated by double strand sequencing.

LC4: (SEQ ID NO: 17) MGWSCIILFLVATATGVHSDIVMTQSALSVAVTPGESVSISCRSSKSLLHSSGKTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18)GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGACATCGTGATGACCCAGAGCGCCCTCAGCGTGGCCGTGACCCCCGGCGAGAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGCCTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCCGGCCAGAGCCCCCAGCTGCTGATCTACCGCATGAGCAACCTGGCCAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGAAGATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGCTGAAGCTT LC5: (SEQ ID NO: 19)MGWSCIILFLVATATGVHSDIVMTQSALSVAVTPGESVSISCRSSKSLLHSSGKTYLYWFLQRPGQSPQLLIYRLSNLASGVPDRFSGSGSGTAFTLKISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 20)GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGACATCGTGATGACCCAGAGCGCCCTCAGCGTGGCCGTGACCCCCGGCGAGAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGCCTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCCGGCCAGAGCCCCCAGCTGCTGATCTACCGCCTGAGCAACCTGGCCAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGAAGATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGCTGAAGCTT LC6: (SEQ ID NO: 21)MGWSCIILFLVATATGVHSDIVMTQSALSVAVTPGESVSISCRSSKSLLHSSGKTYLYWFLQRPGQSPQLLIYRLSSLASGVPDRFSGSGSGTAFTLKISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22)GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGACATCGTGATGACCCAGAGCGCCCTCAGCGTGGCCGTGACCCCCGGCGAGAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGCCTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCCGGCCAGAGCCCCCAGCTGCTGATCTACCGCCTGAGCAGCCTGGCCAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGAAGATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGCTGAAGCTT HC3: (SEQ ID NO: 23)MGWSCIILFLVATATGVHSQVQLQESGPGLVAPSESLSITCTVSGFSLIDYGVNWIRQPPGKGLEWLGVIWGDGTTYYNPSLKSRLSISKDNSKSQVFLKMNSLTAADTAMYYCARIVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG(SEQ ID NO: 24) GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCGAGAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAGCCTGATCGACTACGGCGTGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCGACGGCACCACCTACTACAACCCCAGCCTGAAGAGCCGCCTGAGCATCTCCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGACCGCCGCCGACACCGCCATGTACTACTGCGCCCGCATCGTGTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCGAGTCCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGTCCAAGTACGGCCCTCCTTGCCCTTCCTGCCCTGCCCCTGAGTTCCTGGGCGGACCTAGCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTTCAATTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGTAAGGTCTCCAACAAGGGCCTGCCCTCCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCTCAGGTGTACACCCTGCCTCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAGGCTGACCGTGGACAAGTCCCGGTGGCAGGAGGGCAACGTCTTTTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGT CTCTGGGCTGAAGCTT

Example 15 Humanization

The V_(L) and V_(H) sequences of 16D7 were blasted and the closesthomologues for the variable light chain are 1MH5, 1MJJ and 1MJU (J MolBiol 332:423-435, 2003), with equivalent similarity scores. 1MJU wasretained as a template because of high accuracy of the crystalstructure, which had been determined down to 1.22 Å resolution. Theclosest homologue for the heavy chain was found to be 1FNS (Nat StructBiol 7:881-884, 2000). The structures, 1MJU and 1FNS, were used to buildup a homology model of the variable domains which was subsequentlyenergy minimized using standard procedures. A molecular dynamic (MD)calculation of a 3D homology model of 16D7 was subsequently performedfor 1.7 nanoseconds in Generalized Born implicit solvent (see Gallicchio& Levy, J Comput Chem 2004, 25:479-499).

The MD starts by an initialization of the velocities from a Gaussiandistribution at 298.15° K, followed by an equilibration period of 300ps. During the MD, all bonds are constrained using the SHAKE algorithm(see Barth. Et al., J Comp Chem, 1995, 16:1192-1209), the time step was1 femtosecond (fs), and the simulation, based on the Verlet integrationalgorithm, was run in the canonical NVT (number of particles, volume andtemperature) ensemble at a temperature of 298.15° K. During theproduction period, 1,700 snapshots were then stored, one every 1 ps. The1,700 conformations of the murine antibody constitute the ensemble onwhich the following analysis was performed to identify the most flexibleresidues. The Scientific Vector Language (SVL), available within the MOEmolecular modeling environment, (Molecular Operating Environment (MOE),Chemical Computing Group, Quebec, Canada) was used to code the followinganalysis. First, each snapshot, N, was optimally superposed onto itspredecessor, the snapshot N−1, to control the overall rotational andtranslational motions which occur during the MD modeling andcalculation. The superposition was obtained by minimizing the Root MeanSquare Distance (RMSD) between all pairs of corresponding atoms from thetwo snapshots. Only the heavy atoms of the antibody backbone wereconsidered in the superposition exercise. Using the same superpositionmethod, each snapshot then was superposed onto the medoïd snapshot. Themedoïd snapshot is the antibody conformation with the Cartesiancoordinates the closest from the average coordinates of allconformations.

For each of the antibody residue i, the RMSD between the heavy atoms ofthe conformation j and a medoïd reference conformation k werecalculated. The RMSD has the following formula:

${{RMSDj} = \sqrt{\frac{\sum\limits_{l = 1}^{m}\left( d_{lk} \right)^{2}}{m}}},$with d_(lk) defined as the Euclidean distance expressed in Angstroms (Å)between the heavy atom 1 of the residue j and its counterpart of themedoïd reference conformation k. For the pairwise association of heavyatoms 1, the symmetry of the side-chain heavy atoms for the amino acids,Asp, Lcu, Val, Glu, Arg, Phe and Tyr, also was considered. The referenceconformation k varies from one residue to another, and corresponds tothe medoïd conformation k with the closest Euclidean distance to theaverage coordinates of all conformations of the studied residue i. Then,for each residue i, a distribution of 1,700 RMSD values, which reflectsthe variation of coordinates of the residue i in the course of the MD,was obtained. By aggregating all the RMSD values of all the residues ofthe studied antibody, a global distribution of all RMSDs was obtained.The global distribution of all RMSD then was used as a referencedistribution. If the residue i is highly flexible, then a statisticaltest was performed to decide whether the observed mean RMSD of residuei, m_(i), was significantly higher than the global mean RMSD for allresidues, m_(g). As the sample is large, e.g. 1,700 for the analysis ofclone 16D7, a one-tailed Z-test (see Dorofeev & Grant, “Statistics forreal-life sample surveys. Non-simple-random samples and weighted data”2006. Cambridge University Press) with the null-hypothesis H₀ being that“the observed m_(i) is lower than the global m_(g)” was used tocalculate the statistic, Z_(i) according to the formula:

${{Zi} = \frac{\left( {m_{i} - m_{global}} \right)}{\sqrt{\frac{{sd}_{i}}{n}}}},$where m_(i) is the mean RMSD calculated from the RMSD distribution ofresidue i, m_(g) is the mean RMSD calculated from the global RMSDdistribution, sd_(i) is the standard deviation calculated from the RMSDdistribution of residue i and n is the sample size, i.e. n=1,700 for theanalysis of the 16D7 clone. The calculated Zi was then compared to thecumulative probabilities of the standard normal distribution to assess a99.9% level of significance of the alternative hypothesis, i.e., that“the observed in, is higher than the global m_(g)”. That corresponded toa Zi≧3.08. The Zi statistic, which can be viewed as a flexibility score,is not correlated with either the molecular weight or the number ofheavy atoms of the antibody residue (r²=0.014 and 0.0009, respectivelywhen analyzing the 16D7 anti-CXCR5 model MD).

The set of flexible residues for the light chain include the followingresidues (sequential numbering): D1, T14, P15, R16, E17, Q47, D65, S72R79, R82, E86, K108, E110 and K112; and for the heavy chain include thefollowing residues: Q1, V2, Q3, L11, S15, Q16, S61, A62, K64, S65, R70,D72, Q75, K81, M82, N83, Q86, Q103, 5110, A111, A112 and K113. Theflexible portions of 16D7 were compared to the corresponding positionsof human antibody sequences in the September 2005 version of theImMunoGeneTics Database website.

Those residues which display a significantly high flexible score and afew flanking residues that preserve the 3D structures of the flexibleresidues were retained for the search.

The human antibody variable region with the most identical flexibleresidues, with special considerations given to positions that comewithin 5.0 Å of a CDR, was chosen to replace the murine the 16D7antibody variable region flexible residues. The resulting humanizedsequences were blasted for sequence similarity in theUniProtKB/SwissProt database providing confidence that reasonableassumptions had been made. All sequences showed a high degree ofsimilarity to a number of human antibodies. In addition, none of thesequences contains any known B-cell or T-cell epitope listed in the IEDBdatabase.

The best sequence match in the IEDB for the light chain (LC1, LC2 andLC3) was KPGQPPRLLIYDASNRATGIPA (SEQ ID NO:25), which covers CDR2 buthas significant residue difference as typified by a 56% sequenceidentity obtained from a BLAST search within the IEDB database.

The best match in the IEDB for the heavy chain (HC1 and HC2) wasTDDTAMYYCAR1 (SEQ ID NO:26) which is located before the start of theCDR3. The sequence exhibits 61% sequence identity with the peptideSEDSALYYCARD (SEQ ID NO:27), making it unlikely to be a potential humanT cell epitope (J Exp Med (1995) 181, 1540)

Original sequences of murine 16D7 variable domains are:

light chain (CDRs underlined): (SEQ ID NO: 28)DIVMTQAAPSVAVTPRESVSISCRSSKSLLHSSGKTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLE IK; and heavy chain (CDRs underlined):(SEQ ID NO: 29) QVQLKESGPGLVAPSQSLSITCTVSGFSLIDYGVNWIRQPPGKGLEWLGVIWGDGTTYYNSALKSRLSIRKDNSQSQVFLKMNSLQTDDTAMYYCARIVYWGQGTLVTVS A.

Two versions for the heavy chain (HC1 & HC2) and three versions weresuggested for the light chain (LC1, LC2 and LC3). Both versions of theheavy chain are derived from AF262096/AAF79987 and AB063657/BAC01285.1,respectively. The two sequences exhibit equivalent similarity score, butwere kept because the sets of residues to mutate appear relativelydifferent and display different physico-chemical properties. The LC1sequence is derived from BAC01682/AB064054.1 and there are only tworesidues to be mutated. LC2 and LC3 are variants of LC1 that aresuggested to address potential problematic residues in the light chain:an exposed methionine (M51) mutated to Lcu (versions 3 and 4) and onepotential deamidation site (version 4) where the asparagine, N53, ischanged to a serine residue. Not all combinations were retained but fourcover most of the key points to be addressed. Kabat numbering is used.

TABLE 2 Light Light Chain Chain Ver- Ver- Ver- Ver- (Sequential (Kabatsion1 sion 2 sion3 sion 4 numbering) numbering) (LC1) (LC1) (LC2) (LC3)Arg16 Arg16 Gly Gly Gly Gly Glu17 Glu17 Ala Ala Ala Ala Met56 Met51 MetMet Leu Leu Asn58 Asn53 Asn Asn Asn Scr In total 2 2 3 4 for VL muta-muta- muta- muta- tions tions tions tions Heavy Heavy Chain Chain (HC1)(HC2) (HC1) (HC1) Gln1 Gln1 Gln Glu Gln Gln Ser15 Ser15 Ser Gly Ser SerGln16 Gln16 Glu Gly Glu Glu Ser61 Ser61 Pro Ala Pro Pro Ala62 Ala62 SerPro Ser Ser Scr65 Scr65 Scr Gly Scr Scr Arg70 Arg70 Ser Ser Ser SerGln75 Gln75 Lys Lys Lys Lys Lys81 Lys81 Lys Gln Lys Lys Met82 Met82 ValMet Val Val Asn83 Asn82A Thr Asn Thr Thr Gln86 Gln83 Thr Lys Thr ThrAla111 Ala113 Ala Ser Ala Ala In total 8 11 8 8 for VH muta- muta- muta-muta- tions tions tions tions

Mutations introduced through the humanization of the variable chains arein lowercase and underlined and CDRs are underlined. The constantdomains are not included.

LC1: (SEQ ID NO: 30) DIVMTQAAPSVAVTPgaSVSISCRSSKSLLHSSGKTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLE IK LC2: (SEQ ID NO: 31)DIVMTQAAPSVAVTPgaSVS ISCRSSKSLLHSSGKTYLYWFLQRPGQSPQLLIYRlSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIK LC3: (SEQ ID NO: 32)DIVMTQAAPSVAVTPgaSVSISCRSSKSLLHSSGKTYLYWFLQRPGQSPQLLIYRlSsLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIK HC1: (SEQ ID NO: 33)QVQLKESGPGLVAPSeSLSITCTVSGFSLIDYGVNWIRQPPGKGLEWLGVIWGDGTTYYNpsLKSRLSIsKDNSkSQVFLKvtSLtTDD TAMYYCARIVYWGQGTLVTVSAHC2: (SEQ ID NO: 34) eVQLKESGPGLVAPggSLSITCTVSGFSLIDYGVNWIRQPPGKGLEWLGVIWGDGTTYYNapLKgRLSIsKDNSkSQVFLqMNSLkTDD TAMYYCARIVYWGQGTLVTVSs

The sequences of the chimeric constructs are as follows:

Chimeric LC Sequence (SEQ ID NO: 35)MGWSCIILFLVATATGVHSDIVMTQAAPSVAVTPRESVSISCRSSKSLLHSSGKTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 36)GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGACATCGTGATGACCCAGGCCGCCCCCAGCGTGGCCGTGACCCCCCGCGAGAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGCCTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCCGGCCAGAGCCCCCAGCTGCTGATCTACCGCATGAGCAACCTGGCCAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGCGCATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGCTGAAGCTT Chimeric HC Sequence(SEQ ID NO: 37) MGWSCIILFLVATATGVHSQVQLKESGPGLVAPSQSLSITCTVSGFSLIDYGVNWIRQPPGKGLEWLGVIWGDGTTYYNSALKSRLSIRKDNSQSQVFLKMNSLQTDDTAMYYCARIVYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG(SEQ ID NO: 38) GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGTGCAGCTGAAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAGCCTGATCGACTACGGCGTGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCGACGGCACCACCTACTACAACAGCGCCCTGAAGAGCCGCCTGAGCATCCGCAAGGACAACAGCCAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATGTACTACTGCGCCCGCATCGTGTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGCCAGCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCGAGTCCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGTCCAAGTACGGCCCTCCTTGCCCTTCCTGCCCTGCCCCTGAGTTCCTGGGCGGACCTAGCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTTCAATTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGTAAGGTCTCCAACAAGGGCCTGCCCTCCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCTCAGGTGTACACCCTGCCTCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAGGCTGACCGTGGACAAGTCCCGGTGGCAGGAGGGCAACGTCTTTTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGT CTCTGGGCTGAAGCTTHumanized VL Sequences LC1: (SEQ ID NO: 39)MGWSCIILFLVATATGVHSDIVMTQAAPSVAVTPGASVSISCRSSKSLLHSSGKTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 40)GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGACATCGTGATGACCCAGGCCGCCCCCAGCGTGGCCGTGACCCCCGGCGCCAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGCCTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCCGGCCAGAGCCCCCAGCTGCTGATCTACCGCATGAGCAACCTGGCCAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGCGCATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGCTGAAGCTT LC2: (SEQ ID NO: 41)MGWSCIILFLVATATGVHSDIVMTQAAPSVAVTPGASVSISCRSSKSLLHSSGKTYLYWFLQRPGQSPQLLIYRLSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42)GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGACATCGTGATGACCCAGGCCGCCCCCAGCGTGGCCGTGACCCCCGGCGCCAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGCCTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCCGGCCAGAGCCCCCAGCTGCTGATCTACCGCCTGAGCAACCTGGCCAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGCGCATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGCTGAAGCTT LC3: (SEQ ID NO: 43)MGWSCIILFLVATATGVHSDIVMTQAAPSVAVTPGASVSISCRSSKSLLHSSGKTYLYWFLQRPGQSPQLLIYRLSSLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 44)GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGACATCGTGATGACCCAGGCCGCCCCCAGCGTGGCCGTGACCCCCGGCGCCAGCGTGAGCATCAGCTGCCGCAGCAGCAAGAGCCTGCTGCACAGCAGCGGCAAGACCTACCTGTACTGGTTCCTGCAGCGCCCCGGCCAGAGCCCCCAGCTGCTGATCTACCGCCTGAGCAGCCTGGCCAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGCCTTCACCCTGCGCATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCCGCTCCTTCCGTGTTCATCTTCCCTCCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTGTGTCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAGTCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGCTGAAGCTT Humanized VH SequencesHC1: (SEQ ID NO: 45) MGWSCIILFLVATATGVHSQVQLKESGPGLVAPSESLSITCTVSGFSLIDYGVNWIRQPPGKGLEWLGVIWGDGTTYYNPSLKSRLSISKDNSKSQVFLKVTSLTTDDTAMYYCARIVYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG(SEQ ID NO: 46) GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGTGCAGCTGAAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCGAGAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAGCCTGATCGACTACGGCGTGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCGACGGCACCACCTACTACAACCCCAGCCTGAAGAGCCGCCTGAGCATCAGCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGGTGACCAGCCTGACCACCGACGACACCGCCATGTACTACTGCGCCCGCATCGTGTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGCCAGCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCGAGTCCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGTCCAAGTACGGCCCTCCTTGCCCTTCCTGCCCTGCCCCTGAGTTCCTGGGCGGACCTAGCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTTCAATTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGTAAGGTCTCCAACAAGGGCCTGCCCTCCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCTCAGGTGTACACCCTGCCTCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAGGCTGACCGTGGACAAGTCCCGGTGGCAGGAGGGCAACGTCTTTTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGT CTCTGGGCTGAAGCTT HC2:(SEQ ID NO: 47) MGWSCIILFLVATATGVHSEVQLKESGPGLVAPGGSLSITCTVSGFSLIDYGVNWIRQPPGKGLEWLGVIWGDGTTYYNAPLKGRLSISKDNSKSQVFLQMNSLKTDDTAMYYCARIVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG(SEQ ID NO: 48) GCTAGCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGAGGTGCAGCTGAAGGAGAGCGGCCCCGGCCTGGTGGCCCCCGGCGGCAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAGCCTGATCGACTACGGCGTGAACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCGACGGCACCACCTACTACAACGCCCCCCTGAAGGGCCGCCTGAGCATCAGCAAGGACAACAGCAAGAGCCAGGTGTTCCTGCAGATGAACAGCCTGAAGACCGACGACACCGCCATGTACTACTGCGCCCGCATCGTGTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCGAGTCCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGTCCAAGTACGGCCCTCCTTGCCCTTCCTGCCCTGCCCCTGAGTTCCTGGGCGGACCTAGCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTTCAATTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGTAAGGTCTCCAACAAGGGCCTGCCCTCCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCTCAGGTGTACACCCTGCCTCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAGGCTGACCGTGGACAAGTCCCGGTGGCAGGAGGGCAACGTCTTTTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGT CTCTGGGCTGAAGCTT

Example 16 Humanization Based on Molecular Dynamic Trajectories

The V_(L) and V_(H) sequences of 16D7 were blasted in the protein database (PDB) (Berman et al., Nucleic Acids Research, 2000, 28:235-242) andthe closest homologues for the variable light chain are 1MH5, 1MJJ and1MJU (J Mol Biol 332:423-435, 2003), with equivalent similarity scores.1MJU was retained as a template because of high accuracy of the crystalstructure, which had been determined up to 1.22 Å resolution. Theclosest homologue for the heavy chain was found to be 1FNS (Nat StructBiol 7:881-884, 2000). The structures, 1MJU and 1FNS, were used to buildup a homology model of the variable domains which was subsequentlyenergy minimized using standard procedures.

A molecular dynamic (MD) simulation of the 3D homology model of 16D7 wassubsequently performed for 1.1 nanosecond (ns) in Generalized Bornimplicit solvent (see Gallicchio & Levy, J Comput Chem 2004,25:479-499). The MD simulation starts by an initialization of thevelocities from a Gaussian distribution at 500 K, followed by anequilibration period of 200 picoseconds (ps). During the MD simulation,all bonds are constrained using the SHAKE algorithm (see Barth. et al.,J Comp Chem, 1995, 16:1192-1209), the time step was 1 femtosecond (fs),and the simulation, based on the Veda integration algorithm, was run inthe canonical NVT (number of particles, volume and temperature) ensembleat a temperature of 500 K. This simulation is done with harmonicconstraints applied to the backbone atoms. Ten diverse conformations areextracted, one every 100 ps, during the last 1 ns of this firstsimulation.

These 10 diverse conformations are then used as 10 diverse startingpoints to run 10 molecular dynamic simulations, without constraints onthe backbone, at a 300 K temperature for 2.3 ns in Generalized Bornimplicit solvent. Each MD simulation starts by an initialization of thevelocities from a Gaussian distribution at 298.15 K, followed by anequilibration period of 300 ps. All bonds are constrained using theSHAKE algorithm (see Barth. et al., J Comp Chem, 1995, 16:1192-1209),the time step was 1 fs, and the simulation, based on the Verletintegration algorithm, was run in the canonical NVT (number ofparticles, volume and temperature) ensemble at a temperature of 298.15K. During the production period, 2,000 snapshots were then stored, oneevery 1 ps. The Scientific Vector Language (SVL), available within theMOE molecular modeling environment, (Molecular Operating Environment(MOE), Chemical Computing Group, Quebec, Canada) was used to code thefollowing post-treatment protocol.

First, each snapshot, N, was optimally superposed onto its predecessor,the snapshot N−1, to discard the overall rotational and translationalmotions which occur during the MD modeling and calculation. Thesuperposition was obtained by minimizing the Root Mean Square Distance(RMSD) between all pairs of corresponding atoms from the two snapshots.Only the heavy atoms of the antibody backbone were considered in thesuperposition operation. Using the same superposition method, eachsnapshot then was superposed onto the medoïd snapshot. The medoïdsnapshot is the antibody conformation with the Cartesian coordinates theclosest from the average coordinates of all conformations. For each ofthe 10 MD simulations, the last 2,000 conformations are used toquantify, for each amino acid of the murine antibody, the deviation ofthe atomic positions with respect to a medoïd conformation of theamino-acid. For each of the antibody residue i, the RMSD between theheavy atoms of the conformation j and a medoïd reference conformation kwere calculated. The RMSD has the following formula:

${{RMSDj} = \sqrt{\frac{\sum\limits_{l = 1}^{m}\left( d_{lk} \right)^{2}}{m}}},$with d_(lk) defined as the Euclidean distance expressed in Angstroms (Å)between the heavy atom l of the residue j and its counterpart of themedoïd reference conformation k. For the pair wise association of heavyatoms l, the symmetry of the side chain heavy atoms for the amino acids,Asp, Leu, Val, Glu, Arg, Phe and Tyr, also was considered. The referenceconformation k varies from one residue to another, and corresponds tothe medoïd conformation k with the closest Euclidean distance to theaverage coordinates of all conformations of the studied residue i.

The humanizing mutations are found by determining which human antibodygerm line is the most similar to the murine antibody in terms of theirmost flexible amino acids. To do so, the motions of the 60 most flexibleamino acids of the murine antibody, during the 20 ns (10×2 ns) ofmolecular dynamic simulation, are compared to the motions of thecorresponding amino acids of 49 homology models of human antibody germlines, for each of which 10 molecular dynamic simulations have been runusing the same protocol. The 49 3D homology models of human antibodygerm lines were built by systematically combining the 7 most frequenthuman light chain (vκ1, vκ2, vκ3, vκ4, vλ1, vλ2, vλ3) and the 7 mostfrequent heavy chains (vh1a, vh1b, vh2, vh3, vh4, vh5, vh6) (NucleicAcids Research, 2005, Vol. 33, Database issue D593-D597).

The 60 most flexible amino acids exclude any amino acid in the CDR, andits immediate vicinity, i.e. amino acid with an α carbon at a distanceof less than 5 Å to any α carbon of CDR amino acids as seen in the 3Dhomology model.

The flexibility is quantified by comparing the RMSD (Fi) of a givenamino acid (i) to its medoïd conformation as defined previously,averaged over 10 molecular dynamic simulations, to the RMSD (Fm) of allamino acids of the murine antibody, averaged over the same 10 moleculardynamic simulations. An amino acid is considered flexible enough topotentially interact with the T-cell receptors, and trigger animmune-response, if the flexibility score Zi, defined as Zi=(Fi−Fm)/Fm,is above 0.15.

Using this molecular dynamic averaged flexibility estimation protocol,23 amino acids have been considered as flexible in the variable regionof the murine 16D7 antibody, excluding the CDR region and its immediatevicinity thereof.

The set of flexible residues for the light chain include the followingresidues (sequential numbering): R16, E17, R44, G46, Q47, S48, R79, R82,E84, E86, and E110; and for the heavy chain include the followingresidues: K5, P41, G42, K43, K64, R70, D72, N73, S74, Q75, Q86, andQ103.

The quadridimensional similarity of the murine antibody to the 49 humangerm line homology models is quantified by sampling the positions ofspecific atoms of the 60 flexible amino acids, using all picosecondsnapshots of the 10 molecular dynamic simulations, by means of a uniquetridimensional cubic grid. This grid has a 1 Å resolution. Thetridimensional grid is made of 445740 points and has been initializedusing the tridimensional structure of a human antibody crystallographicmodel based on antibody, 8FAB (Biochem 30:3739-3748, 1991). The 8FABmodel is also used to position all picosecond snapshot conformations ofan antibody which are sampled in the tridimensional grid. For thispurpose, the medoïd conformation of the molecular dynamic of theantibody is superposed onto the 8FAB model. This superposition consistsof aligning the moments of inertia of the 2 models, followed by theminimization of the scalar distances between the α carbons of bothmodels. All the remaining conformations of the molecular dynamicsimulation are superposed onto the medoïd conformation using the samemethod.

Two types of sampling are done which result in two similarities(electrostatic similarity and lipophilic similarity), for a pair ofantibodies being compared. These two similarities are then added toobtain the total similarity. The electrostatic sampling considers allatoms of the amino acid side chain. The value in one point, x, of thegrid is obtained by applying a tridimensional Gaussian function f(x)weighted with the atomic partial charge as described in the Amber99force field (Cieplak et al.; J. Comp. Chem. 2001, 22:1048-1057). Thef(x) function is applied on the 3 Cartesian coordinate axis using thefollowing formula:

${{f(x)} = {\left( {s\sqrt{2\;\pi}} \right)^{- 3} \times {\exp\left( \frac{- \left( {x - u} \right)^{2}}{2\; s^{2}} \right)}}},$x and u being, respectively, the Cartesian coordinates of a grid pointand a sampled amino-acid atom, and s=r/1.6 (r=covalence radius of theatom). The sampling is repeated for all conformations of the amino acidand the obtained results are averaged at all points of thetridimensional grid. The lipophilic sampling considers only thelipophilic atoms of the amino acid side-chain. The value at one point ofthe grid is calculated with the same Gaussian function f(x) withoutweighting. As a result, the two ensembles of picosecond snapshotconformations from the molecular dynamic simulations, of the twoantibodies being compared are sampled by the same tridimensional grid.The electrostatic similarity (sim-elec), between antibody a and antibodyb, can be calculated with the following formula:

${{sim} - {elec}} = {\frac{\sum\limits_{i = 1}^{445740}\left( {{{x_{i}^{a} + x_{i}^{b}}} - {{x_{i}^{a} - x_{i}^{b}}}} \right)}{\sum\limits_{i = 1}^{445740}\left( {{x_{i}^{a} + x_{i}^{b}}} \right)}.}$The lipophilic similarity is calculated with the same formula applied tothe data generated by the lipophilic sampling previously described.

The human germ line model vλ2-vh4 displays the highest quadridimensionalsimilarity (total similarity=50%) of its 60 most flexible amino acidswith respect to the 60 most flexible amino acids of the murine antibody16D7. The human germ line model vλ2-vh4 has thus been used to replacethe murine antibody 16D7 flexible residues. Beforehand, the amino acidsof the two sequences were aligned according to the optimal superpositionof the α carbons of the corresponding 3D homology models. Unwantedmotifs were searched for in the resulting humanized sequences using ablast search, as previously described in paragraph 41 above. Inaddition, known B-cell or T-cell epitopes were searched for in theresulting humanized sequences using the IEDB database as described inparagraph 44, supra.

The best sequence match in the IEDB for the light chain (LC7) wasPGKAPQLLIYRMSNL (SEQ ID NO:52), which covers CDR2. The sequence exhibits73% sequence identity with the peptide PGKAPKLLIYAASSL (SEQ ID NO:53)which shows binding to HLA-DRB1 0404* but has not been demonstrated tobe immunogenic in man (J Immunol (1995) 155, 5655).

The best match in the IEDB for the heavy chain (HC4 and HC5) wasSLIDYGVNWIRQPPG (SEQ ID NO:54) which covers CDR1 but has significantresidue difference as typified by a 40% sequence identity obtained froma BLAST search within the IEDB database.

Two versions for the heavy chain (HC4 & HC5) and one version for thelight chain (LC7) were obtained. Both versions of the heavy chain arederived from the human germ line model vλ2-vh4. HC5 is a variant of HC4with an additional mutation to address a potential problematic residue:one potential deamidation site where the asparagine, N60, is changedinto a proline residue.

Mutations introduced through the humanization of the variable chains arein lowercase and underlined and CDR's are underlined. The constantdomains are not included.

LC7: (SEQ ID NO: 55) DIVMTQAAPSVAVTPgqSVSISCRSSKSLLHSSGKTYLYWFLQhPGkaPQLLIYRMSNLASGVPDRFSGSGSGTAFTLtISgVqAEDVGVYYCMQHLE YPYTFGGGTKLEIK HC4:(SEQ ID NO: 56) QVQLqESGPGLVAPSQSLSITCTVSGFSLIDYGVNWIRQPPGKGLEWLGVIWGDGTTYYNSALKSRLSIsKDtSkSQVFLKMNSLtTDDTAMYYCARIVYWGQGTLVTVSAAK HC5: (SEQ ID NO: 57)QVQLqESGPGLVAPSQSLSITCTVSGFSLIDYGVNWIRQPPGKGLEWLGVIWGDGTTYYpSALKSRLSIsKDtSkSQVFLKMNSLtTDDTAMYYCARIVYWGQGTLVTVSAAK

Light Chain (Sequential numbering) (LC7) ARG16 GLY GLU17 GLN ARG44 HISGLN47 LYS SER48 ALA ARG79 THR ARG82 GLY GLU84 GLN In total 8 for VLmutations Heavy Chain (HC4) (HC5) LYS5 GLN GLN ASN60 ASN PRO ARG70 SERSER ASN73 THR THR GLN75 LYS LYS GLN86 THR THR In total 5 6 for VHmutations mutations

Example 17 Pharmacokinetics

The study is conducted with a suitable number of animals, (for example,4 in a study; 2 for single dose and 2 for repeat dose, 5 doses weekly)healthy, purpose-bred male Cynomolgus monkeys weighing between 2.0 and5.4 kg and ranging from 2 to 7 years of age. The animals can beallocated to two treatment groups, one receiving control IgG4 antibodiesand one group receiving humanized 16D7. Monkeys in each group areadministered a single intravenous bolus dose for example, 2.5-10 mg/kgin a dose volume of 2-3 mL/kg, or 5 weekly doses, i.v. Blood samples arecollected at various time points after each dose administration andprocessed to plasma. The plasma samples are analyzed for concentrationof total IgG4 and CXCR5 mAbs using an ELISA.

Example 18 Comparative Studies

Some of the instant antibodies were compared to commercially availableantibodies in side-by-side experiments. MAB 190, available from R & DSystems, is a mouse mAb. RF82B is a rat anti-human CXCR5 available fromBD. Clone 2C1 is a mouse mAb with a GST tag available from Abnova. Thevarious humanized antibodies described herein were isotyped usingreagents and methods known in the art. For example, those taught hereinhave a κ light chain, many are IgG1, while 46C9, 68D3 and H28 IgG2a.Most of the antibodies bind to the amino terminal end of CXCR5, andseveral of the antibodies compete with each other for binding to thesame epitope or region.

The BD antibody binds poorly to human PBMCs.

While the antibodies generally did not bind to cynomolgus cells, 14C9,19H5, H28, 54G6, 56H6 and 79B7 of the instant invention did.

16D7 was found to be of higher affinity, at least 10-fold, than thecommercial antibodies, and has an off rate 100 times better than theother antibodies.

Example 19 Scale-Up

Each monoclonal antibody variant was produced in suspension-cultivatedHEK293 FS™ cells by transient transfection of two expression plasmidsencoding the heavy or the light chain complexed with 293Fectin™(Invitrogen). Secreted proteins were harvested eight dayspost-transfection and centrifuged. Proteins were purified by affinitychromatography on Protein A (ProSepvA, Millipore) after elution from thecolumn with 25 mM citrate pH 3, 0.15 M NaCl buffer. The monoclonalantibodies were formulated in PBS and 0.22 μm filtered. Proteinconcentration was determined by measurement of absorbance at 280 nm.Each batch was analyzed by SDS-PAGE (Nupagc Bistris/MES-SDS 10%) underreducing and non-reducing conditions to determine the purity and themolecular weight of each subunit and of the monomer. Each protein lotwas also analyzed by gel filtration (Tricorn 10/300 GL Superdex 200) todetermine the homogeneity of the monomer and the presence of highmolecular weight species.

From 240 mL cultures, a total of 30 to 40 mg of eight 16D7 variantmonoclonal antibodies was available and of appropriate quality forsubsequent in vitro and in vivo tests.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed:
 1. A method of making a humanized antibody or fragmentthereof, comprising: (a) identifying a human variable region homologousto a variable region of a non-human antibody; (b) building a homologymodel of the variable region of the non-human antibody using humanvariable region identified in step (a) and running molecular dynamicsimulations to obtain molecular dynamic trajectory of the variableregion of the non-human antibody; (c) analyzing the molecular dynamictrajectory of the variable region of the non-human antibody anddetermining amino acid flexibility scores to identify amino acids in thevariable region of the non-human antibody which are flexible and aminoacids that flank the flexible residue; (d) building homology models ofhuman antibody germ lines and running molecular dynamic simulations toobtain molecular dynamic trajectories of the variable region of thehuman antibody germ lines; (e) analyzing the molecular dynamictrajectories of the variable region of the human antibody germ lines anddetermining amino acid flexibility scores to identify amino acids in thevariable region of the human antibody germ lines which are flexible andamino acids that flank the flexible residue; (f) identifying the humanvariable region of the human antibody germ lines that displays moleculardynamic trajectory most similar to the molecular dynamic trajectory ofthe variable region of the non-human antibody and identifying humanizingmutations by comparing the flexibility scores for the most flexibleamino acids of steps (c) and (e); (g) replacing the identified aminoacids of step (c) with the identified amino acids of step (f) to producea humanized variable region; (h) confirming that the humanized variableregion of step (g) resembles a human antibody by comparing the humanizedvariable region molecular dynamic trajectory with the molecular dynamictrajectory of the variable region of the human antibody germ lines; and(i) recombinantly constructing a humanized variable region by joiningthe humanized variable region of step (g) with a human sequence to yielda humanized antibody or fragment thereof.
 2. The method of claim 1,wherein step (g) comprises not replacing amino acids more than 5 Å froma complementarity determining region.
 3. The method of claim 1, furthercomprising confirming that the humanized variable region of step (g)resembles a human antibody by comparing the humanized variable regionsequence with the sequences of a collection of human antibody sequences.4. The method of claim 1, wherein the humanized variable region of step(g) does not comprise a B cell epitope or a T cell epitope.
 5. A methodof making a humanized antibody or fragment thereof that specificallybinds to the extracellular domain of human CXCR5, comprising: (a)identifying a human variable region homologous to a variable region of anon-human CXCR5 antibody; (b) building a homology model of the variableregion of the non-human CXCR5 antibody using human variable regionidentified in step (a) and running molecular dynamic simulations toobtain molecular dynamic trajectory of the variable region of thenon-human antibody; (c) analyzing the molecular dynamic trajectory ofthe variable region of the non-human antibody and determining amino acidflexibility scores to identify amino acids in the variable region of thenon-human antibody which are flexible and amino acids that flank theflexible residue; (d) building homology models of human antibody germlines and running molecular dynamic simulations to obtain moleculardynamic trajectories of the variable region of the human antibody germlines; (e) analyzing the molecular dynamic trajectories of the variableregion of the human antibody germ lines and determining amino acidflexibility scores to identify amino acids in the variable region of thehuman antibody germ lines which are flexible and amino acids that flankthe flexible residue; (f) identifying the human variable region of thehuman antibody germ lines that displays molecular dynamic trajectorymost similar to the molecular dynamic trajectory of the variable regionof the non-human antibody and identifying humanizing mutations bycomparing the flexibility scores for the most flexible amino acids ofsteps (c) and (e); (g) replacing the identified amino acids of step (c)with the identified amino acids of step (f) to produce a humanizedvariable region; h) confirming that the humanized variable region ofstep (g) resembles a human antibody by comparing the humanized variableregion molecular dynamic trajectory with the molecular dynamictrajectory of the variable region of the human antibody germ lines; and(i) recombinantly constructing a humanized variable region by joiningthe humanized variable region of step (g) with a human sequence to yielda humanized antibody or fragment thereof that specifically binds to theextracellular domain of human CXCR5.
 6. The method of claim 5, whereinstep (g) comprises not replacing amino acids more than 5 Å from acomplementarity determining region.
 7. The method of claim 5, furthercomprising confirming that the humanized variable region of step (g)resembles a human antibody by comparing the humanized variable regionsequence with the sequences of a collection of human antibody sequences.8. The method of claim 5, wherein the humanized variable region of step(g) does not comprise a B cell epitope or a T cell epitope.
 9. Themethod of claim 5, wherein the humanized antibody or fragment thereofcomprises: (a) the amino acid sequences of RSSKSLLHSSGKTYLY (SEQ ID NO:58), RMSNLAS (SEQ ID NO: 59), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63); (b) alight chain variable domain comprising the amino acid sequence of SEQ IDNO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and a heavy chain variabledomain comprising the amino acid sequence of SEQ ID NO: 16; (c) theamino acid sequences of RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSNLAS (SEQID NO: 64), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61),VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63); (d) the amino acidsequences of RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSSNLAS (SEQ ID NO: 65),MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ IDNO: 62), and IVY (SEQ ID NO: 63); (e) a variable light chain (VL)comprising the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 19, orSEQ ID NO: 21, and a variable heavy chain (VH) comprising the amino acidsequence of SEQ ID NO: 23; (f) a variable light chain comprising theamino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32,and a variable heavy chain comprising the amino acid sequence of SEQ IDNO: 33 or SEQ ID NO: 34; (g) the amino acid sequences of RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RMSNLA (SEQ ID NO: 66), MQHLEYPYT (SEQ ID NO: 60),GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ IDNO: 63); (h) the amino acid sequences of RSSKSLLHSSGKTYL Y (SEQ ID NO:58), RLSNLA (SEQ ID NO: 67), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63); (i) theamino acid sequences of RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSSLA (SEQ IDNO: 68), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61),VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63); (j) a variable lightchain comprising the amino acid sequence of SEQ ID NO: 35, and avariable heavy chain comprising the amino acid sequence of SEQ ID NO:37; (k) a variable light chain comprising the amino acid sequence of SEQID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 43, and a variable heavy chaincomprising the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 47;(l) a variable light chain comprising the amino acid sequence of SEQ IDNO: 55, and a variable heavy chain comprising the amino acid sequence ofSEQ ID NO: 56 or SEQ ID NO: 57; or (m) the amino acid sequences ofRSSKSLLHSSGKTYLYW (SEQ ID NO: 69), RMSNLA (SEQ ID NO: 66), MQHLEYPYT(SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62),and IVY (SEQ ID NO: 63).
 10. The method of claim 5, wherein thehumanized antibody or fragment thereof comprises a variable light chaincomprising the amino acid sequence of SEQ ID NO: 32, and a variableheavy chain comprising the amino acid sequence of SEQ ID NO:
 33. 11. Themethod of claim 5, wherein the humanized antibody or fragment thereofcomprises the amino acid sequences of RSSKSLLHSSGKTYLY (SEQ ID NO: 58),RLSSLA (SEQ ID NO: 68), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ IDNO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63).